Oligomers and oligomer conjugates

ABSTRACT

The present invention relates to an oligomer conjugate for use in the treatment of a viral disorder. The oligomer conjugate comprises: a) an oligomer capable of modulating a target sequence in HBx and/or HBsAg of Hepatitis B Virus (HBV) to treat said viral disorder; and b) a carrier component capable of delivering the oligomer to the liver which is linked, preferably conjugated, to the oligomer.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional and claims priority to U.S. applicationSer. No. 16/530,765, filed Aug. 2, 2019, which is a continuation andclaims priority to U.S. application Ser. No. 14/714,004, filed May 15,2015, which claims priority benefit of Great Britain Patent ApplicationNo. 1408623.5 having a filing date of May 15, 2014, the entire contentsof which are incorporated herein by reference in its entirety.

SEQUENCE LISTING

This application hereby incorporates by reference the material of theelectronic Sequence Listing filed concurrently herewith. The material inthe electronic Sequence Listing is submitted as a text (.txt) fileentitled “146392033400SeqList.txt” created on May 14, 2015, which has afile size of 286 KB, and is herein incorporated by reference in itsentirety.

FIELD OF INVENTION

The invention relates to the field of oligomer therapeutics, and inparticular to oligomers and oligomer conjugates targeting Hepatitis BVirus (HBV). In particular, the invention relates to the field ofoligomer conjugates therapeutics wherein antisense oligonucleotides areattached to a carrier component. In certain aspects, the inventionrelates to the field of oligomer conjugates therapeutics whereinantisense oligonucleotides are covalently attached to a carriercomponent by means of physiologically labile linkers. More specifically,the present invention relates to oligomers, in particular oligomerconjugates therapeutics, that target HBV mRNA in a cell leading totreatment of the viral disorders.

BACKGROUND

Molecular strategies are being developed to modulate unwanted geneexpression that either directly causes, participates in, or aggravates adisease state. One such strategy involves inhibiting gene expressionwith oligonucleotides complementary in sequence to the messenger RNA ofa deleterious target gene. The messenger RNA strand is a copy of thecoding DNA strand and is therefore, as the DNA strand, called the sensestrand. Oligonucleotides that hybridize to the sense strand are calledantisense oligonucleotides. Binding of these strands to mRNA interfereswith the translation process and consequently with gene expression.

Certain nucleotide-based compounds have been utilized in varioustherapeutic applications. In particular, various oligonucleotides havebeen investigated including single-stranded and double-strandedoligonucleotides, and analogues. To be useful in in vivo applications,an oligonucleotide must have a plethora of properties including theability to penetrate a cell membrane, have good resistance to extra- andintracellular nucleases, have high affinity and specificity for thetarget and preferably have the ability to recruit endogenous enzymessuch as RNAseH, RNAaseIII, RNAseL etc.

A fundamental property of oligonucleotides that underlies many of theirpotential therapeutic applications is their ability to recognize andhybridize specifically to complementary single stranded nucleic acidsemploying either Watson-Crick hydrogen bonding (A-T and G-C) or otherhydrogen bonding schemes such as the Hoogsteen/reverse Hoogsteen mode.Affinity and specificity are properties commonly employed tocharacterize hybridization characteristics of a particularoligonucleotide. Affinity is a measure of the binding strength of theoligonucleotide to its complementary target (expressed as thethermostability (Tm) of the duplex). Each nucleobase pair in the duplexadds to the thermostability and thus affinity increases with increasingsize (number of nucleobases) of the oligonucleotide. Specificity is ameasure of the ability of the oligonucleotide to discriminate between afully complementary and a mismatched target sequence.

Modified nucleic acids are known to be used to improve for examplestabilisation of the oligonucleotides, in particular for the gapmerdesigns, such as when 1 or more modified nucleotides are present ineither or both of the wing regions. Examples of modifications include2′-O-alkyl-RNA units, 2′-amino-DNA units, 2′-fluoro-DNA units, LNAunits, arabino nucleic acid (ANA) units, 2′-fluoro-ANA units, HNA units,INA (intercalating nucleic acid—Christensen, 2002. Nucl. Acids. Res.2002 30: 4918-4925, hereby incorporated by reference) units, 2′MOEunits, ENA (ethylene nucleic acid), UNA (Unlocked Nucleic Acid, Fluiteret al., Mol. Biosyst., 2009, 10, 1039), Tricyclo DNA (R. Steffens & C.J. Leumann, J. Am. Chem. Soc, 1997, 119, 11548-49), cET-LNA (shownherein), and LNA. FIG. 4 presents drawings of some of these analogues.

A particular efficacious modified nucleic acid is referred to as aLocked Nucleic Acids (LNA). LNAs have been reported in the art—forexample see International Patent Application WO 99/14226; P. Nielsen etal, J. Chem. Soc., Perkin Trans. 1, 1997, 3423; P. Nielsen et al., Chem.Commun., 1997, 9, 825; N. K. Christensen et al., J. Am. Chem. Soc.,1998, 120, 5458; A. A. Koshkin et al., J. Org. Chem., 1998, 63, 2778; A.A Koshkin et al. J. Am. Chem. Soc. 1998, 120, 13252-53; Kumar et al.Bioorg, & Med. Chem. Lett., 1998, 8, 2219-2222; and S. Obika et al.,Bioorg. Med. Chem. Lett., 1999, 515. Interestingly, incorporation of LNAunits containing a 2′-0,4′-C-methylene bridge into an oligonucleotidesequence leads to an unprecedented improvement in the hybridizationstability of the modified oligonucleotide (see above and e.g., S. K.Singh et al., Chem. Commun, 1998, 455). Oligonucleotides comprising the2′-0,4′-C-methylene bridge (LNA) units and also the corresponding2′-thio-LNA (thio-LNA), 2′-HN-LNA (amino-LNA), and 2′-N(R)-LNA(amino-R-LNA) analogue, form duplexes with complementary DNA and RNAwith thermal stabilities not previously observed for bi- and tricyclicnucleosides modified oligonucleotides. The increase in Tm permodification varies from +3 to +11° C., and furthermore, the selectivityis also improved. No other DNA analogue has reproducibly shown such highaffinity for nucleic acids.

In a particular aspect, the present invention relates to hepatitis Bvirus (HBV) therapies. Hepatitis B is a viral disease caused by thehepatitis B virus (HBV). It is transmitted parenterally by contaminatedmaterial such as blood and blood products, contaminated needles,sexually and vertically from infected or carrier mothers to theiroffspring. In those areas of the world where the disease is commonvertical transmission at an early age results in a high proportion ofinfected individuals becoming chronic carriers of hepatitis B. It isestimated by the World Health Organization that more than 2 billionpeople have been infected worldwide, with about 4 million acute casesper year, 1 million deaths per year, and 350-400 million chroniccarriers. Approximately 25% of carriers die from chronic hepatitis,cirrhosis, or liver cancer and nearly 75% of chronic carriers are Asian.Hepatitis B virus is the second most significant carcinogen behindtobacco, causing from 60% to 80% of all primary liver cancer. HBV is 100times more contagious than HIV.

The hepatitis B virus (HBV) is an enveloped, partially double-strandedDNA virus. The compact 3.2 kb HBV genome consists of four overlappingopen reading frames (ORF), which encode for the core, polymerase (Pol),envelope and X-proteins. The Pol ORF is the longest and the envelope ORFis located within it, while the X and core ORFs overlap with the PolORF. The lifecycle of HBV has two main events: 1) generation of closedcircular DNA (cccDNA) from relaxed circular (RC DNA), and 2) reversetranscription of pregenomic RNA (pgRNA) to produce RC DNA. Prior to theinfection of host cells, the HBV genome exists within the virion as RCDNA. It has been determined that HBV virions are able to gain entry intohost cells by non-specifically binding to the negatively chargedproteoglycans present on the surface of human hepatocytes and via thespecific binding of HBV surface antigens (HBV sAg). Once the virion hasentered the cell, the viral cores and the encapsidated RC DNA aretransported by host factors, via a nuclear localization signal, into thenucleus through the Impβ/Impa nuclear transport receptors. Inside thenucleus, host DNA repair enzymes convert the RC DNA into cccDNA. cccDNAacts as the template for all viral mRNAs and as such, is responsible forHBV persistence in infected individuals. The transcripts produced fromcccDNA are grouped into two categories; pgRNA and subgenomic RNA.Subgenomic transcripts encode for the three envelope (L, M and S) and Xproteins, and pgRNA encodes for Pre-Core, Core, and Pol proteins.Inhibition of HBV gene expression or HBV RNA synthesis leads to theinhibition of HBV viral replication and antigens production. Forinstance, IFN-α was shown to inhibit HBV replication and viral HBsAgproduction by decreasing the transcription of pregenomic RNA (pgRNA) andsubgenomic RNA from the HBV covalently closed circular DNA (cccDNA)minichromosome. All HBV viral mRNAs are capped and polyadenylated, andthen exported to the cytoplasm for translation. In the cytoplasm, theassembly of new virons is initiated and nascent pgRNA is packaged withviral Pol so that reverse transcription of pgRNA, via a single strandedDNA intermediate, into RC DNA can commence. The mature nucleocapsidscontaining RC DNA are enveloped with cellular lipids and viral L, M, andS proteins and then the infectious HBV particles are then released bybudding at the intracellular membrane. Interestingly, non-infectiousparticles are also produced that greatly outnumber the infectiousvirions. These empty, enveloped particles (L, M and S), are referred toas subviral particles. Importantly, since subviral particles share thesame envelope proteins and as infectious particles, it has been surmisedthat they act as decoys to the host immune system and have been used forHBV vaccines. The S, M, and L envelope proteins are expressed from asingle ORF that contains three different start codons. All threeproteins share a 226aa sequence, the S-domain, at their C-termini. M andL have additional pre-S domains, Pre-S2 and Pre-S2 and Pre-S1,respectively. However, it is the S-domain that has the HBsAg epitope.

Hepatitis B viral infections are a continuing medical problem because,like any rapidly-replicating infectious agent, there are continuingmutations that help some sub-populations of HBV become resistant tocurrent treatment regimens. Currently the recommended therapies forchronic HBV infection by the American Association for the Study of LiverDiseases (AASLD) and the European Association for the Study of the Liver(EASL) include interferon alpha (INFa), pegylated interferon alpha-2a(Peg-IFN2a), entecavir, and tenofovir. However, typical interferontherapy is 48-weeks and results in serious and unpleasant side effects,and HBeAg seroconversion, 24 weeks after therapy has ceased, ranges fromonly 27-36%. Seroconversion of HBsAg is even lower—only 3% observedimmediately after treatment ceases, with an increase to upwards of 12%after 5 years.

The secretion of antiviral cytokines in response to HBV infection by thehepatocytes and/or the intra-hepatic immune cells plays a central rolein the viral clearance of infected liver. However, chronically infectedpatients only display a weak immune response due to various escapestrategies adopted by the virus to counteract the host cell recognitionsystems and the subsequent antiviral responses.

Many observations showed that several HBV viral proteins couldcounteract the initial host cellular response by interfering with theviral recognition signaling system and subsequently the interferon (IFN)antiviral activity. Among these, the excessive secretion of HBV emptysubviral particles (SVPs, HBsAg) may participate to the maintenance ofthe immunological tolerant state observed in chronically infectedpatients (CHB). In particular, SVPs could contribute to the absence ofantigen presentation by dendritic cells together with the lack ofHBV-specific T cell immune activation to enable viral persistence. HBsAgquantification is a significant biomarker to predict the infectionoutcome; however the achievement of HBsAg seroconversion is rarelyobserved in chronically infected patients. The reduction of the SVP andHBsAg burden is thought as a pathway to recover anti-viral immunefunction and the seroconversion to HBsAg-negative after antiviraltherapy can be seen as an indication as a functional cure and remainsthe ultimate goal of therapy. Therefore, targeting HBV gene expressionleading to reduction of HBsAg together with HBV DNA levels in CHBpatients may significantly improve CHB patient immune reactivation andremission.

The nucleoside and nucleotide therapies entecavir and tenofovir aresuccessful at reducing viral load, but the rates of HBeAg seroconversionand HBsAg loss are even lower than those obtained using IFNa therapy.Other similar therapies, including lamivudine (3TC), telbivudine (LdT),and adefovir are also used, but for nucleoside/nucleotide therapies ingeneral, the emergence of resistance limits therapeutic efficacy.

U.S. Pat. No. 8,598,334 and WO 2012/145697 mention the use of antisenseoligonucleotides to target HBV.

The control of viral infection needs a tight surveillance of the hostinnate immune system which could respond within minutes to hours afterinfection to impact on the initial growth of the virus and limit thedevelopment of a chronic and persistent infection. Despite the availablecurrent treatments based on IFN and nucleos(t)ide analogues, theHepatitis B virus (HBV) infection remains a major health problemworldwide which concerns an estimated 350 million chronic carriers whohave a higher risk of liver cirrhosis and hepatocellular carcinoma.

There is a need for new anti-viral therapies, in particular anti-HBVtherapies. There is also a need to have a therapeutic strategy thatenables one to target different types of HBV genotypes.

SUMMARY OF INVENTION

The present invention relates to oligomer conjugates, uses thereof,methods using same, that are suitable for use in medicine, such as thetreatment of a viral disorder.

In particular, the present invention provides an oligomer or an oligomerconjugate—that is suitable for use in the treatment of a viraldisorder—wherein said oligomer or said oligomer of the oligomerconjugate is capable of modulating a target sequence of Hepatitis BVirus (HBV), preferably HBx or HBsAg of HBV. In certain embodiments acarrier component is conjugated to the oligomer.

The invention provides an oligomer or an oligomer conjugate as hereindefined wherein the oligomer or oligomer component of the oligomerconjugate comprises at least 6, preferably at least 7, preferably atleast 8, preferably at least 9, preferably at least 10 units, preferablyat least 11 units, preferably at least 12 units, preferably at least 13units, preferably at least 14 units, preferably at least 15 units,preferably at least 16 units that are at least 80% identical, preferablyat least 85% identical, preferably at least 90% identical, preferably atleast 91% identical, preferably at least 92% identical, preferably atleast 93% identical, preferably at least 94% identical, preferably atleast 95% identical, preferably at least 96% identical, preferably atleast 97% identical, preferably at least 98% identical, preferably atleast 99% identical, preferably identical to a region corresponding to aHBV HBx gene or HBsAg gene or to the reverse complement of a targetregion of a nucleic acid which encodes a HBV HBx or HBV HBsAg.

The invention provides an oligomer or an oligomer conjugate as hereindefined wherein the oligomer or oligomer component of the oligomerconjugate comprises at least 6, preferably at least 7, preferably atleast 8, preferably at least 9, preferably at least 10 units that areidentical to a region corresponding to a HBV HBx gene or HBsAg gene orto the reverse complement of a target region of a nucleic acid whichencodes a HBV HBx or HBV HBsAg.

For certain embodiments, the invention provides an oligomer or anoligomer conjugate as herein defined wherein the oligomer or oligomercomponent of the oligomer conjugate comprises less than 20 units, suchas less than 19 units, such as less than 18 units, such as less than 17units, such as 16 or less units.

For certain embodiments, the invention provides an oligomer or anoligomer conjugate as herein defined wherein the oligomer or oligomercomponent of the oligomer conjugate comprises 15 units or 16 units.

The invention provides a conjugate comprising the oligomer according tothe invention, and at least one non-nucleotide or non-polynucleotidemoiety covalently attached to said oligomer.

The invention provides a pharmaceutical composition comprising theoligomer or the conjugate according to the invention, and apharmaceutically acceptable diluent (such as water or saline), carrier,salt or adjuvant.

The invention provides the oligomer or the conjugate according to theinvention for use as a medicament, such as for the treatment of a viraldisorder.

The invention provides the use of an oligomer or the conjugate accordingto the invention for the manufacture of a medicament for the treatmentof a viral disorder.

The invention provides a method of treating a viral disorder, saidmethod comprising administering an effective amount of, an oligomer, anoligomer conjugate or a pharmaceutical composition according to theinvention, to an animal suffering from, or likely to suffer from a viraldisorder (such as an animal suffering from or susceptible to the diseaseor disorder).

The invention provides a method of treating a viral disorder, saidmethod comprising administering an effective amount of, an oligomer, anoligomer conjugate or a pharmaceutical composition according to theinvention, to a non-human animal suffering from, or likely to sufferfrom a viral disorder (such as a non-human animal suffering from orsusceptible to the disease or disorder).

The invention provides a method of treating a viral disorder, saidmethod comprising administering an effective amount of, an oligomer, anoligomer conjugate or a pharmaceutical composition according to theinvention, to a human patient suffering from, or likely to suffer from aviral disorder (such as a human patient suffering from or susceptible tothe disease or disorder).

Also disclosed are methods of treating an animal (a non-human animal ora human) suspected of having, or susceptible to, a disease or condition,associated with expression, or over-expression of HBx or HBsAg byadministering to the non-human animal or human a therapeutically orprophylactically effective amount of one or more of the oligomers,conjugates or pharmaceutical compositions of the invention. Further,methods of using oligomers for the inhibition of expression of HBx orHBsAg, and for treatment of diseases associated with activity of HBx orHBsAg are provided.

The invention further provides a pharmaceutical system comprising apharmaceutical composition according to the invention and an additionalpharmaceutical entity/therapeutic entity. The additional pharmaceuticalentity may be an oligomer or oligomer conjugate according to the presentinvention. The additional pharmaceutical entity may be an oligomer oroligomer conjugate capable of modulating a target sequence in HBV whichis not within HBx or HBsAg. For example, at least one oligomer oroligomer conjugate may be capable of modulating a target sequence withinthe gene or mRNA for HBV HBsAg, HBeAg, or DNA polymerase.

In one embodiment, a plurality of oligomers or conjugates according tothe invention are administered to a subject in need of treatment. Theoligomers or conjugates may administered with additionalpharmaceutical/therapeutic agents.

In one embodiment, the disease or disorder or condition is associatedwith overexpression of HBx or HBsAg.

The invention provides for methods for modulating the expression of HBxor HBsAg in a cell or a tissue, the method comprising the step ofcontacting the cell or tissue, in vitro or in vivo, with an effectiveamount of one or more oligomers, conjugates, or pharmaceuticalcompositions thereof, to effect modulation of expression of HBx orHBsAg.

The invention provides for methods of inhibiting (e.g., bydown-regulating) the expression of HBx in a cell or a tissue, the methodcomprising the step of contacting the cell or tissue, in vitro or invivo, with an effective amount of one or more oligomers, conjugates, orpharmaceutical compositions thereof, to effect down-regulation ofexpression of HBx or HBsAg.

The invention provides for a method for the inhibition of HBx or HBsAgin a cell which is expressing HBx or HBsAg, said method comprisingadministering an oligomer, or a conjugate according to the invention tosaid cell so as to affect the inhibition of HBx or HBsAg in said cell.

Further provided are methods of down-regulating the expression of HBx orHBsAg in cells or tissues comprising contacting said cells or tissues,in vitro or in vivo, with an effective amount of one or more of theoligomers, oligomer conjugates or compositions of the invention.

Aspects of the present invention are now provided.

In one aspect, the present invention provides an oligomer conjugate foruse in the treatment of a viral disorder, wherein said oligomerconjugate comprises:

-   -   a) at least one first oligomer region capable of modulating a        target sequence of Hepatitis B Virus (HBV), preferably HBx or        HBsAg of HBV, to treat said viral disorder; and    -   b) a carrier component. Preferably, for delivering said first        oligomer to the liver.

In one aspect, the present invention provides an oligomer conjugatesuitable for the treatment of a viral disorder, wherein said oligomerconjugate comprises:

-   -   a) at least one first oligomer region capable of modulating a        target sequence of Hepatitis B Virus (HBV), preferably HBx or        HBsAg of HBV, to treat said viral disorder; and    -   b) a carrier component. Preferably, for delivering said first        oligomer to the liver.

In one aspect, the present invention provides a composition suitable forthe treatment of a viral disorder, wherein said composition comprises anoligomer conjugate and at least one additional differentoligonucleotide; wherein said oligomer conjugate comprises:

-   -   a) at least one first oligomer region capable of modulating a        target sequence of Hepatitis B Virus (HBV), preferably HBx or        HBsAg of HBV, to treat said viral disorder; and    -   b) a carrier component. Preferably, for delivering said first        oligomer to the liver.

In one aspect, the present invention provides an oligomer or theoligomer component of the oligomer conjugate that hybridize to a targetsequence selected from the group consisting of any one or more ofpositions: 1530 to 1598; 1264-1278 and 670 to 706 of SEQ ID NO: 3.

In one aspect, the present invention provides an oligomer based on acore motif selected from the group consisting of any one or more of:

(SEQ ID NO: 13) GCGTAAAGAGAGG; (SEQ ID NO: 11) GCGTAAAGAGAGGT;(SEQ ID NO: 20) AGCGAAGTGCACACG; (SEQ ID NO: 26) AGGTGAAGCGAAGTG;(SEQ ID NO 18) AGCGAAGTGCACACGG; (SEQ ID NO: 7) CGAACCACTGAACA;(SEQ ID NO 4) GAACCACTGAACAAA; (SEQ ID NO 5) CGAACCACTGAACAAA;(SEQ ID NO 6) CGAACCACTGAACAA; (SEQ ID NO 8) CGAACCACTGAAC (SEQ ID NO 9)CCGCAGTATGGATCG (SEQ ID NO: 10) CGCAGTATGGATC; (SEQ ID NO 12)CGCGTAAAGAGAGGT; (SEQ ID NO 14) AGAAGGCACAGACGG; (SEQ ID NO 15)GAGAAGGCACAGACGG (SEQ ID NO 16) GAAGTGCACACGG; (SEQ ID NO 17)GCGAAGTGCACACGG; (SEQ ID NO 19) CGAAGTGCACACG; (SEQ ID NO 27)AGGTGAAGCGAAGT; and (SEQ ID NO: 852) TAGTAAACTGAGCCA,which is capable of modulating a target sequence in HBx or HBsAg of HBVto treat a viral disorder.

In one aspect, the present invention provides a composition suitable forthe treatment of a viral disorder, wherein said composition comprises anoligomer and at least one additional different oligonucleotide; whereinsaid oligomer comprises at least one first oligomer region capable ofmodulating a target sequence of Hepatitis B Virus (HBV), preferably HBxor HBsAg of HBV, to treat said viral disorder.

In one aspect, the present invention provides a method for treating aviral disorder, said method comprising administering to a subject inneed of treatment an effective amount of an oligomer conjugate accordingto the invention.

In one aspect, the present invention provides a method for treating aviral disorder, said method comprising administering to a subject inneed of treatment an effective amount of a composition according to theinvention.

In one aspect, the present invention provides a method for treating aviral disorder, said method comprising administering to a subject inneed of treatment an effective amount of an oligomer according to theinvention.

In one aspect, the present invention provides a pharmaceuticalcomposition comprising an oligomer conjugate according to the invention;and one or more pharmaceutically acceptable diluents, carriers, salts oradjuvants.

In one aspect, the present invention provides a pharmaceuticalcomposition comprising a composition according to the invention; and oneor more pharmaceutically acceptable diluents, carriers, salts oradjuvants.

In one aspect, the present invention provides a pharmaceuticalcomposition comprising an oligomer according to the invention; and oneor more pharmaceutically acceptable diluents, carriers, salts oradjuvants.

In one aspect, the present invention provides a pharmaceutical systemcomprising a pharmaceutical composition according to the invention andan additional pharmaceutical entity.

In one aspect, the present invention provides a motif selected from thegroup consisting of any one or more of:

(SEQ ID NO: 13) GCGTAAAGAGAGG; (SEQ ID NO: 11) GCGTAAAGAGAGGT;(SEQ ID NO: 20) AGCGAAGTGCACACG; (SEQ ID NO: 26) AGGTGAAGCGAAGTG;(SEQ ID NO 18) AGCGAAGTGCACACGG;

for constructing an oligomer conjugate according to the invention.

In one aspect, the present invention provides a motif selected from thegroup consisting of any one or more of: NO: 13) GCGTAAAGAGAGGT (SEQ IDNO: 11) and CGCGTAAAGAGAGGT (SEQ ID NO 12) for constructing acomposition according to the invention.

In one aspect, the present invention provides a motif selected from thegroup consisting of any one or more of: AGCGAAGTGCACACG (SEQ ID NO: 20);AGGTGAAGCGAAGTG (SEQ ID NO: 26); AGCGAAGTGCACACGG (SEQ ID NO 18);GAAGTGCACACGG (SEQ ID NO 16); GCGAAGTGCACACGG (SEQ ID NO 17);CGAAGTGCACACG (SEQ ID NO 19) and AGGTGAAGCGAAGT (SEQ ID NO 27) forconstructing an oligomer according to the invention.

In one aspect, the present invention provides a method of manufacturingan oligomer conjugate according to the invention, comprising conjugatingone or more oligomers according to the invention with a carriercomponent according to the invention.

In one aspect, the present invention provides a method of manufacturinga composition according to the invention, comprising admixing anoligomer conjugate according to the invention with a pharmaceuticallyacceptable diluents, carriers, salts or adjuvants.

BRIEF DESCRIPTION OF FIGURES

FIG. 1 : Presents a scheme for a LNA oligomer GalNAc conjugation step.

FIG. 2 : Presents examples of a cholesterol or mono-GalNAc carriercomponents including a C6 linker moiety (region E) which is used to linkthe carrier component to the oligomer (region A or to a physiologicallylabile linker region PL, such as a PO linker). The wavy line representsthe covalent link to the oligomer or region PL.

FIG. 3 : Presents examples of tri-GalNAc carrier components. Conjuagtes1-4 illustrate 4 suitable GalNAc carrier components, and conjugates1a-4a refer to the same carrier components with an additional C6 linkermoiety (region E) which is used to link the carrier component to theoligomer (region A or to a physiologically labile linker, region PL,such as a PO linker). The wavy line represents the covalent link to theoligomer or region PL.

FIG. 4 : Presents examples of oligomer conjugates with trivalent GalNAccarrier components.

FIG. 5 : Presents structures for a series of nucleoside analogues.

FIG. 6 : present the structure of SEQ ID NO: 808

FIG. 7 : present the structure of SEQ ID NO: 814

FIG. 8 : present the structure of SEQ ID NO: 815

FIG. 9 : present the structure of SEQ ID NO: 825

FIG. 10 : present the structure of SEQ ID NO: 826

FIG. 11 : HBsAG reduction of SEQ ID NO: 807 at dose 0.28 mpk (▪), 1.4mg/kg (▴) and 7.1 mpk (▾); SEQ ID NO: 808 at dose 7.1 mg/kg (▪), 1.42mg/kg (▴) and 0.29 mg/kg (▾); SEQ ID NO: 814 at dose 0.252 mg/kg (▴) and1.26 mg/kg (▾), 6.15 mg/kg (♦); SEQ ID NO: 815 at dose 0.3 mg/kg (▪),1.5 mg/kg (▴) and 7.5 mg/kg (▾); SEQ ID NO: 825 at dose 0.3 mg/kg (▴),1.5 mg/kg (▾), and 7.5 mg/kg (♦); SEQ ID NO: 826 at dose 7.1 mg/kg (▪),1.42 mg/kg (▴) and 0.29 mg/kg (▾).

FIG. 12 : HBeAG reduction of SEQ ID NO: 807 at dose 0.28 mg/kg (▪), 1.4mg/kg (▴) and 7.1 mg/kg (▾); SEQ ID NO: 808 at dose 7.1 mg/kg (▪), 1.42mg/kg (▴) and 0.29 mg/kg (▾); SEQ ID NO: 814 at dose 0.252 mg/kg (▴) and1.26 mg/kg (▾), 6.15 mg/kg (♦); SEQ ID NO: 815 at dose 0.3 mg/kg (▪),1.5 mg/kg (▴) and 7.5 mg/kg (▾); SEQ ID NO: 825 at dose 0.3 mg/kg (▴),1.5 mg/kg (▾), and 7.5 mg/kg (♦); SEQ ID NO: 826 at dose 7.1 mg/kg (♦),1.42 mg/kg (▴) and 0.29 mg/kg (▾).

FIG. 13 : HBV DNA reduction of SEQ ID NO: 807 at dose 0.28 mg/kg (▪),1.4 mg/kg (▴) and 7.1 mg/kg (▾); SEQ ID NO: 808 at dose 7.1 mg/kg (▪),1.42 mg/kg (▴) and 0.29 mg/kg (▾); SEQ ID NO: 814 at dose 0.252 mg/kg(▴) and 1.26 mg/kg (▾), 6.15 mg/kg (♦); SEQ ID NO: 815 at dose 0.3 mg/kg(▪), 1.5 mg/kg (▴) and 7.5 mg/kg (▾); SEQ ID NO: 825 at dose 0.3 mg/kg(▴), 1.5 mg/kg (▾), and 7.5 mg/kg (♦); SEQ ID NO: 826 at dose 7.1 mg/kg(♦), 1.42 mg/kg (▴) and 0.29 mg/kg (▾).

FIGS. 14A, 14B and 14C: Presents results from subcutaneous (SC) andintravenous (IV) administration routes of SEQ ID NO: 807 at dose 0.2mg/kg, 1.0 mg/kg and 5.0 mg/kg. FIG. 14A) presents HBsAG reduction. FIG.14B) presents HBeAG reduction. FIG. 14C) presents DNA reduction.

FIGS. 15A, 15B and 15C: Presents results on unconjugated oligomers (▪)(SEQ ID NO:308 and 303) and conjugated oligomers (▾) (SEQ ID NO: 807 and815) tested at equimolar oligomer dosages. FIG. 15A) presents HBsAGreduction. FIG. 15B) presents HBeAG reduction. FIG. 15C) presents DNAreduction.

DEFINITIONS/ELEMENTS OF THE INVENTION

The following presents definitions of terms that apply to all aspects ofthe present invention. These definitions are not mutually exclusive.These definitions also teach additional embodiments regarding allaspects of the present invention.

Oligomer/Oligonucleotide

In the context of the present invention, references to “oligomer” and“oligonucleotide” as used herein also apply to the first oligomer regionand/or the second oligomer region—such as when the first oligomer regionis in the oligomer conjugate or when it is not in the free form (i.e.when not in the oligomer conjugate).

The term “oligomer” refers to a molecule formed by covalent linkage oftwo or more nucleotides (i.e. an oligonucleotide). Therefore as usedherein, the terms “oligomer” and “oligonucleotide” are interchangeableand have identical meaning. Herein, a single nucleotide (unit) may alsobe referred to as a monomer or unit. In some embodiments, the terms“nucleoside”, “nucleotide”, “unit” and “monomer” are usedinterchangeably. It will be recognised that when referring to a sequenceof nucleotides or units, what is referred to is the sequence of bases,such as A, T, G, C or U.

As used herein, the terms “oligomer” and “oligonucleotide” includelinear or circular oligomers of natural and/or modified units orlinkages, including deoxyribonucleosides, ribonucleosides, substitutedand alpha-anomeric forms thereof, peptide nucleic acids (PNA), and thelike, capable of specifically binding to a target polynucleotide by wayof a regular pattern of unit-to-unit interactions, such as Watson-Cricktype of base pairing, Hoogsteen or reverse Hoogsteen types of basepairing, or the like.

The oligonucleotide may be composed of a single region or may becomposed of several regions. The oligonucleotide may be “chimeric”, thatis, composed of different regions. In the context of this invention“chimeric” antisense compounds are antisense compounds, particularlyoligonucleotides, which contain two or more chemical regions, forexample, DNA region(s), RNA region(s), PNA region(s) etc. Each chemicalregion is made up of at least one unit, i.e., a nucleotide in the caseof an oligonucleotide compound. These oligonucleotides typically containat least one region wherein the oligonucleotide is modified in order toexhibit one or more sought properties. The sought properties of theoligonucleotide might be to increased resistance to nucleasedegradation, increased cellular uptake, and/or increased bindingaffinity for the target nucleic acid. Different regions of theoligonucleotide may therefore have different properties. One ore moreregions of the oligonucleotide may serve as a substrate for enzymescapable of cleaving RNA:DNA or RNA:RNA hybrids. There are severalenzymes with such catalytic effect. A method of digesting RNA at aspecific location with an antisense oligonucleotide and an RNase H hasbeen demonstrated by Minshull et al. (Nucleic Acids Research,14:6433-6451 (1986)). Rnase H is a cellular endonuclease which cleavesthe RNA strand of an RNA:DNA duplex. Therefore, activation of RNase Hresults in cleavage of the RNA target. The efficiency of oligonucleotideinhibition of gene expression might therefore be enhanced. Other enzymescapable of cleaving are Rnase L and Rnase P.

Nucleobase

The term “nucleobase” refers to the base moiety of a nucleotide andcovers both naturally occurring a well as non-naturally occurringvariants.

Thus, “nucleobase” covers not only the known purine and pyrimidineheterocycles but also heterocyclic analogues and tautomeres thereof.

Examples of nucleobases include, but are not limited to adenine,guanine, cytosine, thymidine, uracil, xanthine, hypoxanthine,5-methylcytosine, isocytosine, pseudoisocytosine, 5-bromouracil,5-propynyluracil, 6-aminopurine, 2-aminopurine, inosine, diaminopurine,and 2-chloro-6-aminopurine.

In some embodiments, at least one of the nucleobases present in theoligomer is a modified nucleobase selected from the group consisting of5-methylcytosine, isocytosine, pseudoisocytosine, 5-bromouracil,5-propynyluracil, 6-aminopurine, 2-aminopurine, inosine, diaminopurine,and 2-chloro-6-aminopurine.

Units

As used herein, the term “units” or “monomers” typically indicatesnucleoside units linked by phosphodiester bonds or analogs thereof toform oligonucleotides ranging in size from a few monomeric units, e.g.,from about 3-4, to about several hundreds of monomeric units. Analogs ofphosphodiester linkages include: phosphorothioate, phosphorodithioate,methylphosphornates, phosphoroselenoate, phosphoramidate, and the like.

Nucleosides and Nucleoside Analogues

In some embodiments, the terms “nucleoside analogue” and “nucleotideanalogue” are used interchangeably.

The term “nucleotide” as used herein, refers to a glycoside comprising asugar moiety, a base moiety and a covalently linked group (linkagegroup), such as a phosphate or phosphorothioate internucleotide linkagegroup, and covers both naturally occurring nucleotides, such as DNA orRNA, and non-naturally occurring nucleotides comprising modified sugarand/or base moieties, which are also referred to as “nucleotideanalogues” herein. Herein, a single nucleotide (unit) may also bereferred to as a monomer or nucleic acid unit.

In field of biochemistry, the term “nucleoside” is commonly used torefer to a glycoside comprising a sugar moiety and a base moiety, andmay therefore be used when referring to the nucleotide units, which arecovalently linked by the internucleotide linkages between thenucleotides of the oligomer.

In the field of biotechnology, the term “nucleotide” is often used torefer to a nucleic acid monomer or unit, and as such in the context ofan oligonucleotide may refer to the base—such as the “nucleotidesequence”—and typically may refer to the nucleobase sequence (i.e. thepresence of the sugar backbone and internucleoside linkages areimplicit).

Likewise, particularly in the case of oligonucleotides where one or moreof the internucleoside linkage groups are modified, the term“nucleotide” may refer to a “nucleoside” for example the term“nucleotide” may be used, even when specifiying the presence or natureof the linkages between the nucleosides.

As one of ordinary skill in the art would recognise, the 5′ terminalnucleotide of an oligonucleotide does not comprise a 5′ internucleotidelinkage group, although may or may not comprise a 5′ terminal group.

Non-naturally occurring nucleotides include nucleotides which havemodified sugar moieties, such as bicyclic nucleotides or 2′ modifiednucleotides, such as 2′ substituted nucleotides.

“Nucleotide analogues” are variants of natural nucleotides, such as DNAor RNA nucleotides, by virtue of modifications in the sugar and/or basemoieties. Analogues could in principle be merely “silent” or“equivalent” to the natural nucleotides in the context of theoligonucleotide, i.e. have no functional effect on the way theoligonucleotide works to inhibit target gene expression. Such“equivalent” analogues may nevertheless be useful if, for example, theyare easier or cheaper to manufacture, or are more stable to storage ormanufacturing conditions, or represent a tag or label. Preferably,however, the analogues will have a functional effect on the way in whichthe oligomer works to inhibit expression; for example by producingincreased binding affinity to the target and/or increased resistance tointracellular nucleases and/or increased ease of transport into thecell. Specific examples of nucleoside analogues are described by e.g.Freier & Altmann; Nucl. Acid Res., 1997, 25, 4429-4443 and Uhlmann;Curr. Opinion in Drug Development, 2000, 3(2), 293-213, and in Scheme 1presented in FIG. 4 .

Linker/Linker Group Etc.

The terms “linker group”, “linkage group”, “linker”, “linker molecule”or “internucleoside linkage” are intended to mean a group capable ofcovalently coupling together two entities, such as nucleotides. Specificexamples include phosphate groups and phosphorothioate groups. Suchlinkers may contain a spacer molecule covalently attached to one or moreactivated groups or functional groups. Optionally, the functional groupof the linker molecule can be treated with a coupling agent to form anactivated group. Such linkers also include tether molecules as describedherein.

The term “brancher region” is intended to mean a group or region capableof covalently coupling together two or more entities, such asnucleotides or for the generation of carrier component clusters such asgalctose clusters.

Alternatively, “linker groups” and “brancher regions” as describedherein may be used to covalently couple nucleotide regions andnon-nucleotide regions, such a linker group is termed L. For example, alinker group may be used to conjugate an oligomer of the invention to acarrier component described herein. For example, a brancher region maybe used to conjugate an oligomer of the invention to one or more carriercomponents described herein. For example, a brancher region may be usedto conjugate one or more oligomers of the invention to a carriercomponent described herein. For example, a brancher region may be usedto conjugate one or more oligomers of the invention to one or morecarrier components described herein.

Oligomer Conjugate

The term “oligomer conjugate” is intended to indicate a heterogenousmolecule formed by the attachment (“conjugation”), such as by thecovalent attachment, of the oligomer as described herein to a carriercomponent.

The linkage, such as covalent conjugation, may be chemical in nature,such as via a linker group, or genetic in nature for example byrecombinant genetic technology, such as in a fusion protein with forexample a reporter molecule (e.g. green fluorescent protein,β-galactosidase, Histag, etc. Alternatively, the oligomer may beconjugated to the carrier component directly without the need for anytether molecule or linker group.

Carrier Component

As used herein, the term “carrier component” relates to a molecularvehicle which is intended to carry or convey the oligomers of theinvention to their desired location, such as desired anatomicallocation.

Carrier components according to the present invention may be used toenhance the activity, cellular distribution and/or cellular uptake ofthe oligomers.

Any suitable carrier component may be used.

The carrier component may be polynucleotide. However, typically, thecarrier component is a non-nucleotide moiety or a non-polynucleotidemoiety.

Examples of non-nucleotide or non-polynucleotide moieties includemacromolecular agents selected from the group consisting ofcarbohydrates, cell surface receptor ligands, drug substances, hormones,lipophilic substances, polymers, proteins, peptides, toxins (e.g.bacterial toxins), vitamins, viral proteins, or combinations thereof.Typical polymers may be polyethylene glycol and/or polypropylene glycol(PPG).

In some embodiments, the carrier component may be an amino acid, aprotein, a peptide or polypeptide. Typically proteins may be enzymes,serum proteins (e.g. human serum albumin (HSA), transferrin orglycoproteins), receptors, antibodies or antibody derivatives thereoflike single-chain variable fragments, bispecific antibodies, tribodiesetc designed to bind a desired target antigen.

Examples of peptide carrier components are poly(L-lysine), thatsignificantly increases cell penetration, and the antennapedia transportpeptide. Such conjugates are described by Lemaitre et al, “Specificantiviral activity of a poly(L-lysine)-conjugatedoligodeoxyribonucleotide sequence complementary to vesicular stomatitisvirus N protein mRNA initiation site,” Proc. Natl. Acad. Sci. USA,84:648-652, 1987; U.S. Pat. Nos. 6,166,089 and 6,086,900. The procedurein the above publication requires that the 3′-terminal nucleotide be aribonucleotide. The resulting aldehyde groups are then randomly coupledto the epsilon-amino groups of lysine residues of poly(L-lysine) bySchiff base formation, and then reduced with sodium cyanoborohydride.This procedure converts the 3′-terminal ribose ring into morpholinestructure antisense oligomers. The peptide segment can also besynthesized by strategies which are compatible with DNA/RNA synthesise.g. Mmt/Fmoc strategies. In that case the peptide can be synthesizeddirectly before or after the oligonucleotide segment. Also methods existto prepare the peptide oligonucleotide conjugate post synthetically,e.g., by formation of a disulfide bridge.

In some embodiments, the conjugate moiety may be or comprise alipophilic conjugate moiety. Lipophilic conjugate moieties may beselected from the group consisting of sterols, stanols, steroids,polycyclic aromatic groups, aliphatic groups, lipids, phospholipids,lipophilic alcohols, fatty acids and fatty esters. In some embodiments,the conjugate moiety comprises cholesterol.

The carrier component may be or comprise a pharmacokinetic modulator,such as a lipophilic or hydrophobic moieties. Such moieties aredisclosed within the context of siRNA conjugates in WO2012/082046. Thehydrophobic moiety may comprise a C8-C36 fatty acid, which may besaturated or un-saturated. In some embodiments, C10, C12, C14, C16, C18,C20, C22, C24, C26, C28, C30, C32 and C34 fatty acids may be used. Thehydrophobic group may have 16 or more carbon atoms. Exemplary suitablehydrophobic groups may be selected from the group comprising: sterol,cholesterol, palmitoyl, hexadec-8-enoyl, oleyl, (9E,12E)-octadeca-9,12-dienoyl, dioctanoyl, and C16-C20 acyl. According toWO2012/082046, hydrophobic groups having fewer than 16 carbon atoms areless effective in enhancing polynucleotide targeting, but they may beused in multiple copies (e.g. 2×, such as 2×C8 or C10, C12 or C14) toenhance efficacy. Pharmacokinetic modulators useful as polynucleotidetargeting moieties may be selected from the group consisting of:cholesterol, alkyl group, alkenyl group, alkynyl group, aryl group,aralkyl group, aralkenyl group, and aralkynyl group, each of which maybe linear, branched, or cyclic. Pharmacokinetic modulators arepreferably hydrocarbons, containing only carbon and hydrogen atoms.However, substitutions or heteroatoms which maintain hydrophobicity, forexample fluorine, may be permitted.

WO2007/031091 provides examples of other suitable ligands and carriercomponents, which are hereby incorporated by reference.

In some embodiments, the carrier component is or comprises acarbohydrate moiety. Carbohydrate conjugate moieties include, but is notlimited to, galactose, lactose, n-acetylgalactosamine, mannose andmannose-6-phosphate. Carbohydrate conjugates may be used to enhancedelivery or activity in a range of tissues, such as liver and/or muscle.See, for example, EP1495769, WO99/65925, Yang et al., Bioconjug Chem(2009) 20(2): 213-21. Zatsepin & Oretskaya Chem Biodivers. (2004) 1(10):1401-17.

In addition, the oligomer may further comprise one or more additionalconjugate moieties, of which lipophilic or hydrophobic moieties areparticularly interesting. These may for example, act as pharmacokineticmodulators, and may be covalently linked to either the carbohydrateconjugate, a linker linking the carbohydrate conjugate to the oligomeror a linker linking multiple carbohydrate conjugates (multi-valent)conjugates, or to the oligomer, optionally via a linker, such as aphysiologically labile linker.

In some embodiments, the carrier component comprises an asiaglycoproteinreceptor (ASGP-R) targeting moiety with affinity equal to or greaterthan that of galactose. The ASPG-R targeting moiety may be selected fromthe group consisting ofgalactose, galactosamine, N-formyl-galactosamine,N-acetylgalactosamine (GalNAc), N-propionyl-galactosamine,N-n-butanoyl-galactosamine, and N-isobutanoylgalactos-amine. In someembodiments the the asialoglycoprotein receptor targeting conjugatemoiety is mono-valent. In other embodiments the carrier componentcomprises a galactose cluster, such as a di-valent, tri-valent ortetra-valent asialoglycoprotein receptor targeting conjugate moiety(i.e. containing 1, 2, 3 or 4 terminal carbohydrate moieties capable ofbinding to the asialoglycoprotein receptor). In some embodiments, thecarrier component comprises a GalNAc (N-acetylgalactosamine), such as amono-valent, di-valent, tri-valent of tetra-valent GalNAc. GalNAcconjugates may be used to target the compound to the liver. A preferredcarrier component is a N-acetylgalactosamine trimer. GalNAc conjugateshave been used with methylphosphonate and PNA antisense oligonucleotides(e.g. U.S. Pat. No. 5,994,517 and Hangeland et al., Bioconjug Chem. 1995November-December; 6(6):695-701) and siRNAs (e.g. WO2009/126933,WO2012/089352 and WO2012/083046) and with LNA and 2′-MOE modifiednucleosides WO 2014/076196 WO 2014/207232, WO 2014/179620 and WO2014/179627. The GalNAc references and the specific conjugates usedtherein are hereby incorporated by reference. WO2012/083046 disclosessiRNAs with GalNAc conjugate moieties which comprise cleavablepharmacokinetic modulators, which are suitable for use in the presentinvention, the preferred pharmacokinetic modulators are C16 hydrophobicgroups such as palmitoyl, hexadec-8-enoyl, oleyl, (9E,12E)-octadeca-9,12-dienoyl, dioctanoyl, and C16-C20 acyl. TheWO2012/083046 cleavable pharmacokinetic modulators may also becholesterol.

The carrier component may be selected from the group consisting of:galactose, galactosamine, N-formyl-galactosamine, N-acetylgalactosamine,Npropionyl-galactosamine, N-n-butanoyl-galactosamine,N-iso-butanoylgalactos-amine, galactose cluster, andN-acetylgalactosamine trimer and may have a pharmacokinetic modulatorselected from the group consisting of: hydrophobic group having 16 ormore carbon atoms, hydrophobic group having 16-20 carbon atoms,palmitoyl, hexadec-8-enoyl, oleyl, (9E,12E)-octadeca-9,12dienoyl,dioctanoyl, and C16-C20 acyl, and cholesterol. Certain GalNac clustersdisclosed in '046 include: (E)-hexadec-8-enoyl (C16), oleyl (C18),(9,E,12E)-octadeca-9,12-dienoyl (C18), octanoyl (C8), dodececanoyl(C12), C-20 acyl, C24 acyl, dioctanoyl (2xC8). The carriercomponent—pharmacokinetic modulator may be linked to the polynucleotidevia a physiologically labile bond or, e.g. a disulfide bond (PO-linker),or a PEG linker. The invention also relates to the use of phosphodiesterlinkers between the oligomer and the carrier component (suitably arepositioned between the oligomer and the carbohydrate conjugate group).

In one embodiments of the present invention, the oligomer is linked,preferably conjugated, to a carrier component, which may be used todeliver oligomers to the liver of a subject e.g. by increasing thecellular uptake of oligomers.

In a particular embodiment, the carrier component may be GalNAc or aGalNAc cluster. FIGS. 2 and 3 presents some carrier components.

For targeting hepatocytes in liver, a preferred targeting ligand is agalactose cluster.

A galactose cluster comprises a molecule having e.g. comprising two tofour terminal galactose derivatives. As used herein, the term galactosederivative includes both galactose and derivatives of galactose havingaffinity for the ASGP-R equal to or greater than that of galactose. Aterminal galactose derivative is attached to a molecule through its C—Icarbon. The ASGP-R is unique to hepatocytes and binds branchedgalactose-terminal glycoproteins. A preferred galactose cluster hasthree terminal galactosamines or galactosamine derivatives each havingaffinity for the asialoglycoprotein receptor. A more preferred galactosecluster has three terminal N-acetyl-galactosamines. Other terms commonin the art include tri-antennary galactose, tri-valent galactose andgalactose trimer. It is known that tri-antennary galactose derivativeclusters are bound to the ASGP-R with greater affinity than bi-antennaryor mono-antennary galactose derivative structures (Baenziger and Fiete,1980, Cell, 22, 611-620; Connolly et al., 1982, 1. Biol. Chern., 257,939-945). Multivalency is required to achieve nM affinity.

A preferred galactose derivative is an N-acetyl-galactosamine (GalNAc).Other saccharides having affinity for the asialoglycoprotein receptormay be selected from the list comprising: galactosamine,N-n-butanoylgalactosamine, and N-iso-butanoylgalactosamine. Theaffinities of numerous galactose derivatives for the asialoglycoproteinreceptor have been studied (see for example: Jobst, S. T. and Drickamer,K. JB. C. 1996, 271, 6686) or are readily determined using methodstypical in the art.

A galactose cluster may comprise two or preferably three galactosederivatives each linked to a central branch point. The galactosederivatives are attached to the central branch point through the C—Icarbons of the saccharides. The galactose derivative is preferablylinked to the branch point via linkers or spacers. A preferred spacer isa flexible hydrophilic spacer (U.S. Pat. No. 5,885,968; Biessen et al.J. Med. Chern. 1995 Vol. 39 p. 1538-1546). A preferred flexiblehydrophilic spacer is a PEG spacer. A preferred PEG spacer is a PEG3spacer (three ethylene units). The branch point can be any smallmolecule which permits attachment of the three galactose derivatives andfurther permits attachment of the branch point to the oligomer. Eachgalactose derivative (carbohydrate moiety) in a GalNAc cluster (e.g.GalNAc) may be joined to the oligomer via a spacer, such as(poly)ethylene glycol linker (PEG), such as a di, tri, tetra, penta,hexa-ethylene glycol linker. The PEG moiety may form a spacer betweenthe galactose derivative sugar moiety and a peptide (di-lysine is shown)linker. An exemplary branch point group is a di-lysine. A di-lysinemolecule contains three amine groups through which three galactosederivatives may be attached and a carboxyl reactive group through whichthe di-lysine may be attached to the oligomer (se for example FIG. 4 ).

The carbohydrate conjugate (e.g. GalNAc), or carbohydrate-linker moiety(e.g. carbohydrate-PEG moiety) may be covalently joined (linked) to theoligomer via a branch point group such as, an amino acid, or peptide,which suitably comprises two or more amino groups (such as 3, 4, or 5),such as lysine, di-lysine or tri-lysine or tetra-lysine. A tri-lysinemolecule contains four amine groups through which three carbohydrateconjugate groups, such as galactose & derivatives (e.g. GalNAc) and afurther conjugate such as a hydrophobic or lipophilic moiety/group maybe attached and a carboxyl reactive group through which the tri-lysinemay be attached to the oligomer. The further conjugate, such aslipophilic/hyrodphobic moiety may be attached to the lysine residue thatis attached to the oligomer.

In some embodiments, the GalNac cluster comprises a peptide linker, e.g.a Tyr-Asp(Asp) tripeptide or Asp(Asp) dipeptide, which is attached tothe oligomer via a biradical linker, for example the GalNac cluster maycomprise biradical linkers such as those illustrated as Conj 3, 3a, 4and 4a in FIG. 3 .

Alternative brancher molecules may be selected from the from the groupconsisting of1,3-bis-[5-(4,4′-dimethoxytrityloxy)pentylamido]propyl-2-[(2-cyanoethyl)-(N,N-diisopropyl)]phosphoramidite (Glen Research Catalogue Number: 10-1920-xx),tris-2,2,2-[3-(4,4′-dimethoxytrityloxy)propyloxymethyl]ethyl-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite(Glen Research Catalogue Number: 10-1922-xx),tris-2,2,2-[3-(4,4′-dimethoxytrityloxy)propyloxymethyl]methyleneoxypropyl-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramiditeand1-[5-(4,4′-dimethoxy-trityloxy)pentylamido]-3-[5-fluorenomethoxy-carbonyl-oxy-pentylamido]-propyl-2-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite(Glen Research Catalogue Number: 10-1925-xx). WO 2014/179620 andEuropean application No. 14188444.5 describes the generation of variousGalNAc conjugate moieties (hereby incorporated by reference). Attachmentof the branch point to oligomer may occur through a linker or spacer. Apreferred spacer is a flexible hydrophilic spacer. A preferred flexiblehydrophilic spacer is a PEG spacer or C6 linker. A preferred PEG spaceris a PEG3 spacer (three ethylene units). In a preferred embodiment thelinker is a physiologically labile linker. The galactose cluster may beattached to the 3′ or 5′ end of the oligomer using methods known in theart. In preferred embodiments the asialoglycoprotein receptor targetingconjugate moiety is linked to the 5′-end of the oligonucleotide.

A preferred galactose cluster comprises three terminal GalNAc moietieslinked via a PEG spacer to a di-lysine brancher molecule (a GalNAccluster). Preferably, the PEG spacer is a 3PEG spacer. Preferred GalNAcclusters are Conj 1, 1a, 2 and 2a. Most preferred is Conj2a (also termedGalNAc2).

Galactose Clusters are Presented in FIG. 3 . Carrier Component Linker

The carrier component may be linked to the first oligomer region, and/orthe second oligomer region, by means of a linker (L). Any suitablelinker may be used.

A carbohydrate conjugate (e.g. GalNAc) may be linked to the oligomer viaa biocleavable linker also termed a physiologically labile linker.

As used herein, a physiologically labile bond is a labile bond that iscleavable under conditions normally encountered or analogous to thoseencountered within a mammalian body (also referred to as aphysiologically labile linker). Physiologically labile linkage groupsare selected such that they undergo a chemical transformation (e.g.,cleavage) when present in certain physiological conditions. Mammalianintracellular conditions include chemical conditions such as pH,temperature, oxidative or reductive conditions or agents, and saltconcentration found in or analogous to those encountered in mammaliancells. Mammalian intracellular conditions also include the presence ofenzymatic activity normally present in a mammalian cell such as fromproteolytic or hydrolytic enzymes

Chemical transformation (cleavage of the labile bond) may be initiatedby the addition of a pharmaceutically acceptable agent to the cell ormay occur spontaneously when a molecule containing the labile bondreaches an appropriate intra- and/or extracellular environment. Forexample, a pH labile bond may be cleaved when the molecule enters anacidified endosome. Thus, a pH labile bond may be considered to be anendosomal cleavable bond. Enzyme cleavable bonds may be cleaved whenexposed to enzymes such as those present in an endosome or lysosome orin the cytoplasm. A disulfide bond may be cleaved when the moleculeenters the more reducing environment of the cell cytoplasm. Thus, adisulfide may be considered to be a cytoplasmic cleavable bond. As usedherein, a pH-labile bond is a labile bond that is selectively brokenunder acidic conditions (pH<7). Such bonds may also be termedendosomally labile bonds, since cell endosomes and lysosomes have a pHless than 7. An example of another physiologically labile linker is adi-lysine as used in the GalNAc clusters in FIG. 3 .

In some embodiments, the oligomer conjugate of the invention comprises aphysiologically labile linker (region PL, also referred to as abiocleavable linker or nuclease susceptible linker), which joins theoligomer (region A) of the invention to the carrier component (or regionC).

For physiologically labile linkers associated with a carrier componentfor targeted delivery it is preferred that, the cleavage rate seen inthe target tissue (for example muscle, liver, kidney or a tumor) isgreater than that found in blood serum. Suitable methods for determiningthe level (%) of cleavage in target tissue versus serum are described inthe “Tissue specific In vitro linker cleavage assay” section. In someembodiments, the physiologically labile linker (also referred to as thebiocleavable linker, or nuclease susceptible linker) in a compound ofthe invention, are at least about 20% cleaved, such as at least about30% cleaved, such as at least about 40% cleaved, such as at least about50% cleaved, such as at least about 60% cleaved, such as at least about70% cleaved, such as at least about 75% cleaved in the “Tissue specificIn vitro linker cleavage assay” in the “Materials and methods” section.In some embodiments, the cleavage (%) in serum, as used in the “Tissuespecific In vitro linker cleavage assay”, is less than about 20%, suchas less than about 10%, such as less than 5%, such as less than about1%.

In some embodiments, the oligomer conjugate of the invention comprisesthree regions: i) a first region (region A), which comprises 10-18contiguous nucleotides; ii) a second region (region PL) which comprisesa physiologically labile linker and iii) a third region (C) whichcomprises a carrier component, wherein the third region is covalentlinked to the second region which is covalently linked to the firstregion.

In some embodiments region A and region PL is covalently linked via aphosphate nucleoside linkage (e.g. phosphodiester, phosphorothioate,phosphodithioate, boranophosphate or methylphosphonate) or a triazolgroup. In some embodiments region PL and region C is covalently linkedvia a phosphate nucleoside linkage (e.g. phosphodiester,phosphorothioate, phosphodithioate, boranophosphate ormethylphosphonate) or a triazol group. In some embodiments region PL andregion C is covalently linked via a second linker such as region Elinkers described below.

In some embodiments, the physiologically labile linker may be situatedeither at the 5′ end and/or the 3′-end of the oligomer (region A). In apreferred embodiment the physiologically labile linker is at the 5′-end.

In some embodiments, the physiologically labile linker is attached atits 3′-end to the 5′-end of region A and the carrier component (regionC) is attached to the 5′-end or the the physiologically labile linker(e.g. PO-linker), optionally via an additional linker region E.

Nuclease Susceptible Linker—Phosphodiester Linker (PO-Linker)

In some embodiments, the physiologically labile linker is susceptible tonuclease(s) which may for example, be expressed in the target cell—andas such, as detailed herein, the linker may be a short region (e.g.1-10) phosphodiester linked nucleosides, such as DNA nucleosides.

In some embodiments, which may be the same or different, thephysiologically labile linker (region PL) is susceptible to S1 nucleasecleavage. Susceptibility to S1 cleavage may be evaluated using the S1nuclease assay described in the “S1 nuclease cleavage assay” section. Insome embodiments, the physiologically labile linker (also referred to asthe physiologically labile linker, or nuclease susceptible linker), suchas region PL, in a compound of the invention, are at least about 30%cleaved, such as at least about 40% cleaved, such as at least about 50%cleaved, such as at least about 60% cleaved, such as at least about 70%cleaved, such as at least about 80% cleaved, such as at least about 90%cleaved, such as at least 95% cleaved after 120 min incubation with S1nuclease as described in the “S1 nuclease cleavage assay in the“Materials and methods” section.

In some embodiments, the physiologically labile linker (region PL) is anuclease susceptible linker, which comprises between 1 and 10nucleosides, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleosides, morepreferably between 2 and 6 nucleosides and most preferably between 2 and4 linked nucleosides. In some embodiments, the nuclease susceptiblelinker is a phosphodiester nucleotide linker, such a linker is alsotermed a PO-linker. In preferred embodiments the nuclease susceptiblelinker (PO-linker) comprises at least one phosphodiester linkednucleoside. Preferably, the nuclease susceptible PO-linker comprises atleast two consecutive phosphodiester linkages, such as at least 3 or 4or 5 consecutive phosphodiester linkages.

In some embodiments, the nucleosides in the PO-linker are (optionallyindependently) selected from the group consisting of DNA and RNA ormodifications thereof which do not interfere with nuclease cleavage.Modifications of DNA and RNA nucleosides which do not interfere withnuclease cleavage may be non-naturally occurring nucleobases. Certainsugar-modified nucleosides may also allow nuclease cleavage such as analpha-L-oxy-LNA. In some embodiments, all the nucleosides of thePO-linker comprise (optionally independently) either a 2′-OH ribosesugar (RNA) or a 2′-H sugar—i.e. RNA or DNA. In some embodiments, thenucleosides of the PO-linker are DNA nucleosides. In some embodiments,at least two consecutive nucleosides of the PO-linker are DNA or RNAnucleosides (such as at least 3 or 4 or 5 consecutive DNA or RNAnucleosides). Preferably the PO-linker consists of between 1 to 5 or 1to 4, such as 2, 3, 4 consecutive phosphodiester linked DNA nucleosides.In preferred embodiments the PO-linker is so short that it does notrecruit RNAseH. In some embodiments, the PO-linker comprises no morethan 3 or no more than 4 consecutive phospodiester linked DNA and/or RNAnucleosides (such as DNA nucleosides).

In some embodiments, the PO-linker is not complementary to the targetnucleic acid sequence or to the oligomer in region A.

In some embodiments, the PO-linker is complementary with the targetnucleic acid sequence. In this respect region A and the PO-linkertogether may form a single contiguous sequence which is complementary tothe target sequence.

In some embodiments, region A and the PO-linker form a single contiguousnucleotide sequence of 10-22, such as 12-20 nucleotides in length. Inthis context region A can be differentiated from the PO-linker in thatit starts with at least one, preferably at least two, modifiednucleosides with increased binding affinity to the target nucleic acid(e.g. LNA or nucleosides with a 2′ substituted sugar moiety) and regionA on its own is capable of modulation of the expression the targetnucleic acid in a relevant cell line. Furthermore, if region A comprisesDNA or RNA nucleosides these are linked with nuclease resistantinternucleoside linkage, such phosphorothioate or boranophosphate.

In some aspects the internucleoside linkage between the first (region A)and the second region (the PO-linker) may be considered part of thesecond region.

In some embodiments, the sequence of bases in the PO-linker is selectedto provide an optimal endonuclease cleavage site, based upon thepredominant endonuclease cleavage enzymes present in the target tissueor cell or sub-cellular compartment. In this respect, by isolating cellextracts from target tissues and non-target tissues, endonucleasecleavage sequences for use in the PO-linker may be selected based upon apreferential cleavage activity in the desired target cell (e.g.liver/hepatocytes) as compared to a non-target cell (e.g. kidney). Inthis respect, the potency of the compound for target down-regulation maybe optimized for the desired tissue/cell.

In some embodiments the PO-linker comprises a dinucleotide of sequenceAA, AT, AC, AG, TA, TT, TC, TG, CA, CT, CC, CG, GA, GT, GC, or GG,wherein C may be 5-methylcytosine, and/or T may be replaced with U.Preferably, the internucleoside linkage is a phosphodiester linkage. Ina preferred embodiment the PO-linker is a CA dinucleotide with at leasttwo phosphodiester linkages (one being to Region A). In some embodimentsthe PO-linker comprises a trinucleotide of sequence AAA, AAT, AAC, AAG,ATA, ATT, ATC, ATG, ACA, ACT, ACC, ACG, AGA, AGT, AGC, AGG, TAA, TAT,TAC, TAG, TTA, TTT, TTC, TAG, TCA, TCT, TCC, TCG, TGA, TGT, TGC, TGG,CAA, CAT, CAC, CAG, CTA, CTG, CTC, CTT, CCA, CCT, CCC, CCG, CGA, CGT,CGC, CGG, GAA, GAT, GAC, CAG, GTA, GTT, GTC, GTG, GCA, GCT, GCC, GCG,GGA, GGT, GGC, and GGG wherein C may be 5-methylcytosine and/or T may bereplaced with U. Preferably, the internucleoside linkages arephosphodiester linkages. In some embodiments the PO-linker comprises atrinucleotide of sequence AAAX, AATX, AACX, AAGX, ATAX, ATTX, ATCX,ATGX, ACAX, ACTX, ACCX, ACGX, AGAX, AGTX, AGCX, AGGX, TAAX, TATX, TACX,TAGX, TTAX, TTTX, TTCX, TAGX, TCAX, TCTX, TCCX, TCGX, TGAX, TGTX, TGCX,TGGX, CAAX, CATX, CACX, CAGX, CTAX, CTGX, CTCX, CTTX, CCAX, CCTX, CCCX,CCGX, CGAX, CGTX, CGCX, CGGX, GAAX, GATX, GACX, CAGX, GTAX, GTTX, GTCX,GTGX, GCAX, GCTX, GCCX, GCGX, GGAX, GGTX, GGCX, and GGGX, wherein X maybe selected from the group consisting of A, T, U, G, C and analoguesthereof, wherein C may be 5-methylcytosine and/or T may be replaced withU. Preferably, the internucleoside linkages are phosphodiester linkages.It will be recognized that when referring to (naturally occurring)nucleobases A, T, U, G, C, these may be substituted with nucleobaseanalogues which function as the equivalent natural nucleobase (e.g. basepair with the complementary nucleoside).

In some embodiments, region PL is a phosphodiester nucleotide linker(PO-linker) covalently attached to a lipophilic conjugate moiety, suchas a lipid, a fatty acid, sterol, such as cholesterol or tocopherol. Insome embodiments, region PL is a phosphodiester nucleotide linker(PO-linker) covalently attached to a asialoglycoprotein receptortargeting moiety, such as a GalNAc carrier component.

The concept of inserting a physiologically labile linker between theoligomer and the carrier component is described in detail in WO2014/076195 (hereby incorporated by reference, in particular FIGS. 3 and4 are incorporated by reference).

Alternative Linkers (Region E)

In some instances linkers are not necessarily physiologically labile butprimarily serves to covalently connect a third region, e.g. a carriercomponent (region C), to an oligomer (region A). Herein these linkersare also termed region E. The oligomer conjugates of the presentinvention can be constructed of the following regional elements A-C/C-A,A-PL-C/C-PL-A, A-PL-E-C/C-E-PL-A, A-E-PL-C/C-PL-E-A or A-E-C/C-E-A.

In some embodiments, the linker E comprises a chain structure or anoligomer of repeating units such as ethylene glycol, amino acid units oramino alkyl groups. The linker E can have at least two functionalities,one for attaching to the oligomer (optionally with a physiologicallylabile linker) and the other for attaching to the carrier component.Example linker functionalities can be electrophilic for reacting withnucleophilic groups on the oligomer or carrier component, ornucleophilic for reacting with electrophilic groups. In someembodiments, linker functionalities include amino, hydroxyl, carboxylicacid, thiol, phosphoramidate, phosphorothioate, phosphate, phosphite,unsaturations (e.g., double or triple bonds), and the like. For example,a carbohydrate carrier component (e.g. GalNAc) may be linked to theoligomer via a linker, such as (poly)ethylene glycol linker (PEG), suchas a di, tri, tetra, penta, hexa-ethylene glycol linker.

In some embodiments the linker (region E) is an amino alkyl, such as aC2-C36 amino alkyl group, including, for example C6 to C12 amino alkylgroups. In a preferred embodiment the linker (region E) is a C6 aminoalkyl group. The amino alkyl group may be added to the oligomer (regionA or region A-L/L-A) as part of standard oligomer synthesis, for exampleusing a (e.g. protected) amino alkyl phosphoramidite. The linkage groupbetween the amino alkyl and the oligomer may for example be aphosphorothioate or a phosphodiester, or one of the other nucleosidelinkage groups referred to herein. The amino alkyl group is covalentlylinked to the 5′ or 3′-end of the oligomer. Commercially available aminoalkyl linkers are for example 3′-Amino-Modifier reagent for linkage atthe 3′-end of the oligomer and for linkage at the 5 ‘-end of an oligomer5’-Amino-Modifier C6 is available. These reagents are available fromGlen Research Corporation (Sterling, Va.). These compounds or similarones were utilized by Krieg, et al, Antisense Research and Development1991, 1, 161 to link fluorescein to the 5′-terminus of an oligomer. Awide variety of further linker groups are known in the art and can beuseful in the attachment of carrier components to oligomers. A review ofmany of the useful linker groups can be found in, for example, AntisenseResearch and Applications, S. T. Crooke and B. Lebleu, Eds., CRC Press,Boca Raton, Fla., 1993, p. 303-350. Other compounds such as acridinehave been attached to the 3′-terminal phosphate group of an oligomer viaa polymethylene linkage (Asseline, et al., Proc. Natl. Acad. Sci. USA1984, 81, 3297). Any of the above groups can be used as a single linker(region E) or in combination with one or more further linkers (regionE-E′ or region E-L or L-E).

Linkers and their use in preparation of conjugates of oligomers areprovided throughout the art such as in WO 96/11205 and WO 98/52614 andU.S. Pat. Nos. 4,948,882; 5,525,465; 5,541,313; 5,545,730; 5,552,538;5,580,731; 5,486,603; 5,608,046; 4,587,044; 4,667,025; 5,254,469;5,245,022; 5,112,963; 5,391,723; 5,510475; 5,512,667; 5,574,142;5,684,142; 5,770,716; 6,096,875; 6,335,432; and 6,335,437, WO2012/083046 each of which is incorporated by reference in its entirety.FIG. 3 present example GalNAc clusters, Conj 1, 2, 3, 4 and Conj1a, 2a,3a and 4a having an optional C6 linker which joins the GalNac cluster tothe oligomer.

Asiaglycoprotein Receptor (ASGP-R) Targeting Moiety

As used herein, the term “asiaglycoprotein receptor (ASGP-R) targetingmoiety” relates to a moiety which interacts with ASGP-R and therebybrings an oligomer into contact with, or into proximity to, a cellexpressing surface ASGP-R.

Conjugate Moiety

In some embodiments, the carrier component is a conjugate moiety.

In addition, one or more conjugate moieties may be attached to theoligomer or the oligomer conjugate in addition to the carrier component.

In some embodiments, one or more conjugate moieties may be attached tothe oligomer of the present invention.

The conjugate moiety may be a non-nucleotide moiety or anon-polynucleotide moiety.

The conjugate moiety may enhance the activity, cellular distribution orcellular uptake of the oligomer of the invention. Such moieties include,but are not limited to, antibodies, polypeptides, lipid moieties such asa cholesterol moiety, cholic acid, a thioether, e.g.Hexyl-s-tritylthiol, a thiocholesterol, an aliphatic chain, e.g.,dodecandiol or undecyl residues, a phospholipids, e.g.,di-hexadecyl-rac-glycerol or triethylammonium1,2-di-o-hexadecyl-rac-glycero-3-h-phosphonate, a polyamine or apolyethylene glycol chain, an adamantane acetic acid, a palmityl moiety,an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety.

The oligomers of the invention may also be conjugated to active drugsubstances for example, aspirin, ibuprofen, a sulfa drug, anantidiabetic, an antibacterial or an antibiotic.

In certain embodiments the conjugated moiety is a sterol, such ascholesterol.

In various embodiments, the conjugate moiety comprises or consists of apositively charged polymer, such as a positively charged peptides of,for example from 1-50, such as 2-20 such as 3-10 amino acid residues inlength, and/or polyalkylene oxide such as polyethylglycol(PEG) orpolypropylene glycol—see WO 2008/034123, hereby incorporated byreference. Suitably the positively charged polymer, such as apolyalkylene oxide may be attached to the oligomer of the invention viaa linker such as the releasable linker described in WO 2008/034123.

The oligomers and oligomer conjugates of the present invention mayinclude—as a conjugate moiety—an appropriate ligand-binding molecule.For example, the oligonucleotides may be conjugated for therapeuticadministration to ligand-binding molecules which recognize cell-surfacemolecules, such as according to International Patent Application WO91/04753. The ligand-binding molecule may comprise, for example, anantibody against a cell surface antigen, an antibody against a cellsurface receptor, a growth factor having a corresponding cell surfacereceptor, an antibody to such a growth factor, or an antibody whichrecognizes a complex of a growth factor and its receptor. Methods forconjugating ligand-binding molecules to oligonucleotides are detailed inWO 91/04753. Further, conjugation methods and methods to improvecellular uptake which may be used are described in the followinginternational patent applications WO 9640961, WO9964449, WO9902673,WO9803533, WO0015265 and U.S. Pat. Nos. 5,856,438 and 5,138,045.

By way of a further example, the conjugate moiety may be a growth factorsuch as transferrin or folate. Transferrin-polylysine-oligonucleotidecomplexes or folate-polylysine-oligonucleotide complexes may be preparedfor uptake by cells expressing high levels of transferrin or folatereceptor. The preparation of transferrin complexes as carrier componentsfacilitating oligonucleotide uptake into cells is described by Wagner etal., Proc. Natl. Acad. Sci. USA 87, 3410-3414 (1990). Cellular deliveryof folate-macromolecule conjugates via folate receptor endocytosis,including delivery of an antisense oligonucleotide, is described by Lowet al., U.S. Pat. No. 5,108,921. Also see, Leamon et al., Proc. Natl.Acad. Sci. 88, 5572 (1991).

Target Nucleic Acid/Target Sequence

In preferred aspects, the terms “target nucleic acid” and “targetsequence”, as used herein refer to the DNA or RNA encoding a HBx orHBsAg polypeptide, such as a sequence contained within any of SEQ ID No.1 or SEQ ID No. 2. The terms “target nucleic acid” and “target sequence”therefore include HBx- or HBsAg-encoding nucleic acids or naturallyoccurring variants thereof, and RNA nucleic acids derived therefrom,preferably mRNA, such as pre-mRNA, although preferably mature mRNA. Insome embodiments, for example when used in research or diagnostics the“target nucleic acid” or “target sequence” may be a cDNA or a syntheticoligonucleotide derived from the above DNA or RNA nucleic acid targets.The oligomer according to the invention is preferably capable ofhybridising to the target nucleic acid.

Identity/Homology

As used herein, the terms “homologous” and “homology” areinterchangeable with the terms “identity” and “identical”.

Corresponding to/Corresponds to

The terms “corresponding to” and “corresponds to” refer to thecomparison between the nucleotide sequence of the oligomer (i.e. thenucleobase or base sequence) or a contiguous nucleotide sequence thereofand the equivalent contiguous nucleotide sequence of a further sequenceselected from either i) a sub-sequence of the reverse complement of thenucleic acid target, such as the mRNA which encodes the target sequenceprotein, such as any sequence within SEQ ID No. 1 or SEQ ID No. 2 or SEQID No. 3 and/or ii) the sequence of any of the specific nucleotidesequences provided herein, or sub-sequence thereof. Nucleotide analoguesare compared directly to their equivalent or corresponding nucleotides.A sequence which corresponds to a further sequence under i) or ii)typically is identical to that sequence over the length of the firstsequence (such as the contiguous nucleotide sequence) or, as describedherein may, in some embodiments, is at least 80% homologous to acorresponding sequence, such as at least 85%, at least 90%, at least91%, at least 92% at least 93%, at least 94%, at least 95%, at least 96%homologous, such as 100% homologous (identical). The percentage sequenceidentity may be calculated by counting the number of aligned bases thatare identical between the 2 sequences, dividing by the total number ofunits in the oligomer, and multiplying by 100.

The terms “corresponding nucleotide analogue” and “correspondingnucleotide” are intended to indicate that the nucleotide in thenucleotide analogue and the naturally occurring nucleotide areidentical. For example, when the 2-deoxyribose unit of the nucleotide islinked to an adenine, the “corresponding nucleotide analogue” contains apentose unit (different from 2-deoxyribose) linked to an adenine.

Complementary

The term, “complementary” means that two sequences are complementarywhen the sequence of one can bind to the sequence of the other in ananti-parallel sense wherein the 3′-end of each sequence binds to the5′-end of the other sequence and each A, T(U), G, and C of one sequenceis then aligned with a T(U), A, C, and G, respectively, of the othersequence. Normally, the complementary sequence of the oligonucleotidehas at least 90%, preferably 95%, most preferably 100%, complementarityto a defined sequence.

In determining the degree of “complementarity” between oligomers of theinvention (or regions thereof) and the target sequence, such as the HBxor HBsAg target sequence, the degree of “complementarity” (also,“homology” or “identity”) is expressed as the percentage identity (orpercentage homology) between the sequence of the oligomer (or regionthereof) and the sequence of the target region (or the reversecomplement of the target region) that best aligns therewith. Thepercentage is calculated by counting the number of aligned bases thatare identical between the 2 sequences, dividing by the total number ofcontiguous units in the oligomer, and multiplying by 100. In such acomparison, if gaps exist, it is preferable that such gaps are merelymismatches rather than areas where the number of units within the gapdiffers between the oligomer of the invention and the target region.

In particular, the term “complementary” means the capacity for pairingbetween nucleobases of a first nucleic acid and a second nucleic acid.Two sequences are complementary when the sequence of one can bind to thesequence of the other in an anti-parallel sense wherein the 3′-end ofeach sequence binds to the 5′-end of the other sequence and eachnucleobase A, T(U), G, and C of one sequence is then aligned with aT(U), A, C, and G, respectively, of the other sequence. In determiningthe degree of complementarity between oligomers of the invention (orregions thereof) and the target sequence, such as the HBx or HBsAgtarget sequence, the degree of complementarity is expressed as thepercentage complementarity between the sequence of the oligomer (orregion thereof) and the sequence of the target region that best alignstherewith. The percentage is calculated by counting the number ofaligned bases that form pairs between the 2 sequences, dividing by thetotal number of contiguous units in the oligomer, and multiplying by100. In such a comparison a nucleobase/nucleotide which does not alignis termed a mismatch. Normally, the complementary sequence of aoligonucleotide of the present invention has at least 80%, preferably85%, preferably 90%, more preferably 95%, most preferably 100%,complementarity to a defined sequence.

The term “complementary sequence” as it refers to a polynucleotidesequence, relates to the base sequence in another nucleic acid moleculeby the base-pairing rules. More particularly, the term or like termrefers to the hybridization or base pairing between nucleotides ornucleic acids, such as, for instance, between the two strands of adouble stranded DNA molecule or between an oligonucleotide primer and aprimer binding site on a single stranded nucleic acid to be sequenced oramplified. Complementary nucleotides are, generally, A and T (or A andU), or C and G. Two single stranded RNA or DNA molecules are said to besubstantially complementary when the nucleotides of one strand,optimally aligned and compared and with appropriate nucleotideinsertions or deletions, pair with at least about 95% of the nucleotidesof the other strand, usually at least about 98%, and more preferablyfrom about 99 to about 100%. Complementary polynucleotide sequences canbe identified by a variety of approaches including use of well-knowncomputer algorithms and software, for example the BLAST program.

Reverse Complement/Reverse Complementary/Reverse Complementarity

The terms “reverse complement”, “reverse complementary” and “reversecomplementarity” as used herein are interchangeable with the terms“complement”, “complementary” and “complementarity”.

Mismatch

The term “mismatch”—that is sometimes referred to as a non-complementarynucleobase—refers to a nucleobase or nucleotide at a given position in afirst nucleic acid that does not make Watson-Crick base paring with thecorresponding nucleobase or nucleotide in a second nucleic acid when thefirst nucleic acid is aligned with thee second nucleic acid. The firstnucleic acid can for example be an oligomer or oligomer conjugateaccording to the invention and the second nucleic acid can for examplebe a target sequence. In the oligomer or the oligomer conjugatecontaining multiple mismatches, the mismatches can either be adjacent toeach other or interspersed.

Naturally Occurring Variant Thereof

The term “naturally occurring variant thereof” refers to variants of thetarget sequence which exist naturally within the defined taxonomicgroup, such as HBV genotypes A-H. Typically, when referring to“naturally occurring variants” of a polynucleotide the term may alsoencompass any allelic variant of the target sequence encoding genomicDNA which are found by chromosomal translocation or duplication, and theRNA, such as mRNA derived therefrom. “Naturally occurring variants” mayalso include variants derived from alternative splicing of the targetsequence mRNA. When referenced to a specific polypeptide sequence, e.g.,the term also includes naturally occurring forms of the protein whichmay therefore be processed, e.g. by co- or post-translationalmodifications, such as signal peptide cleavage, proteolytic cleavage,glycosylation, etc.

Downstream

As used herein, the term “downstream” when used in reference to adirection along a nucleotide sequence means in the direction from the 5′to the 3′ end. Similarly, the term “upstream” means in the directionfrom the 3′ to the 5′ end.

LNA

The term “LNA” refers to a bicyclic nucleoside analogue, known as“Locked Nucleic Acid”. It may refer to an LNA unit, or, when used in thecontext of an “LNA oligonucleotide”, LNA refers to an oligonucleotidecontaining one or more such bicyclic nucleotide analogues. LNAnucleotides are characterised by the presence of a linker group (such asa bridge) between C2′ and C4′ of the ribose sugar ring—for example asshown as the biradical R^(4*)-R^(2*) as described below.

As used herein, the terms “LNA oligonucleotide” and “LNA-modifiedoligonucleotide” include any oligonucleotide either fully or partiallymodified with LNA units. Thus, an LNA-modified oligonucleotide may becomposed entirely of LNA units, or a LNA-modified oligonucleotide maycomprise one LNA unit.

The LNA used in the oligonucleotide compounds of the inventionpreferably has the structure of the general formula I

wherein for all chiral centers, asymmetric groups may be found in eitherR or S orientation;wherein X is selected from —O—, —S—, —N(R^(N*))—, —C(R⁶R^(6*))—, suchas, in some embodiments —O—; B is selected from hydrogen, optionallysubstituted C₁₋₄-alkoxy, optionally substituted C₁₋₄-alkyl, optionallysubstituted C₁₋₄-acyloxy, nucleobases including naturally occurring andnucleobase analogues, DNA intercalators, photochemically active groups,thermochemically active groups, chelating groups, reporter groups, andligands; preferably, B is a nucleobase or nucleobase analogue;

P designates an internucleotide linkage to an adjacent unit, or a5′-terminal group, such internucleotide linkage or 5′-terminal groupoptionally including the substituent R⁵ or equally applicable thesubstituent R^(5*);

P* designates an internucleotide linkage to an adjacent unit, or a3′-terminal group; R^(4*) and R^(2*) together designate a bivalentlinker group consisting of 1-4 groups/atoms selected from—C(R^(a)R^(b))—, —C(R^(a))═C(R^(b))—, —C═N—, —O—, —Si(R^(a))₂—, —S—,—SO₂—, —N(R^(a))—, and >C═Z, wherein Z is selected from —O—, —S—, and—N(R^(a))—, and R^(a) and R^(b) each is independently selected fromhydrogen, optionally substituted C₁₋₁₂-alkyl, optionally substitutedC₂₋₁₂-alkenyl, optionally substituted C₂₋₁₂-alkynyl, hydroxy, optionallysubstituted C₁₋₁₂-alkoxy, C₂₋₁₂-alkoxyalkyl, C₂₋₁₂-alkenyloxy, carboxy,C₁₋₁₂-alkoxycarbonyl, C₁₋₁₂-alkylcarbonyl, formyl, aryl,aryloxy-carbonyl, aryloxy, arylcarbonyl, heteroaryl,heteroaryloxy-carbonyl, heteroaryloxy, heteroarylcarbonyl, amino, mono-and di(C₁₋₆-alkyl)amino, carbamoyl, mono- anddi(C₁₋₆-alkyl)-amino-carbonyl, amino-C₁₋₆-alkyl-aminocarbonyl, mono- anddi(C₁₋₆-alkyl)amino-C₁₋₆-alkyl-aminocarbonyl, C₁₋₆-alkyl-carbonylamino,carbamido, C₁₋₆-alkanoyloxy, sulphono, C₁₋₆-alkylsulphonyloxy, nitro,azido, sulphanyl, C₁₋₆-alkylthio, halogen, DNA intercalators,photochemically active groups, thermochemically active groups, chelatinggroups, reporter groups, and ligands, where aryl and heteroaryl may beoptionally substituted and where two geminal substituents R^(a) andR^(b) together may designate optionally substituted methylene (═CH₂),wherein for all chiral centers, asymmetric groups may be found in eitherR or S orientation, and;

each of the substituents R^(1*), R², R³, R⁵, R^(5*), R⁶ and R^(6*),which are present is independently selected from hydrogen, optionallysubstituted C₁₋₁₂-alkyl, optionally substituted C₂₋₁₂-alkenyl,optionally substituted C₂₋₁₂-alkynyl, hydroxy, C₁₋₁₂-alkoxy,C₂₋₁₂-alkoxyalkyl, C₂₋₁₂-alkenyloxy, carboxy, C₁₋₁₂-alkoxycarbonyl,C₁₋₁₂-alkylcarbonyl, formyl, aryl, aryloxy-carbonyl, aryloxy,arylcarbonyl, heteroaryl, heteroaryloxy-carbonyl, heteroaryloxy,heteroarylcarbonyl, amino, mono- and di(C₁₋₆-alkyl)amino, carbamoyl,mono- and di(C₁₋₆-alkyl)-amino-carbonyl, amino-C₁₋₆-alkyl-aminocarbonyl,mono- and di(C₁₋₆-alkyl)amino-C₁₋₆-alkyl-aminocarbonyl,C₁₋₆-alkyl-carbonylamino, carbamido, C₁₋₆-alkanoyloxy, sulphono,C₁₋₆-alkylsulphonyloxy, nitro, azido, sulphanyl, C₁₋₆-alkylthio,halogen, DNA intercalators, photochemically active groups,thermochemically active groups, chelating groups, reporter groups, andligands, where aryl and heteroaryl may be optionally substituted, andwhere two geminal substituents together may designate oxo, thioxo,imino, or optionally substituted methylene; wherein R^(N) is selectedfrom hydrogen and C₁₋₄-alkyl, and where two adjacent (non-geminal)substituents may designate an additional bond resulting in a doublebond; and R^(N*), when present and not involved in a biradical, isselected from hydrogen and C₁₋₄-alkyl; and basic salts and acid additionsalts thereof. For all chiral centers, asymmetric groups may be found ineither R or S orientation.

In some embodiments, R^(4*) and R^(2*) together designate a biradicalconsisting of functional groups selected from the group consisting ofC(R^(a)R^(b))—C(R^(a)R^(b))—, C(R^(a)R^(b))—O—, C(R^(a)R^(b))—NR^(a)—,C(R^(a)R^(b))—S—, and C(R^(a)R^(b))—C(R^(a)R^(b))—O— wherein each R^(a)and R^(b) may optionally be independently selected. In some embodiments,R^(a) and R^(b) may be, optionally independently selected from the groupconsisting of hydrogen and C₁₋₆alkyl, such as methyl, such as hydrogen.

In some embodiments, R^(4*) and R^(2*) together designate the biradical—O—CH(CH₂OCH₃)-(2′O-methoxyethyl bicyclic nucleic acid—Seth at al.,2010, J. Org. Chem)—in either the R- or S-configuration.

In some embodiments, R^(4*) and R^(2*) together designate the biradical—O—CH(CH₂CH₃)-(2′O-ethyl bicyclic nucleic acid—Seth at al., 2010, J.Org. Chem).—in either the R- or S-configuration.

In some embodiments, R^(4*) and R^(2*) together designate the biradical—O—CH(CH₃)—.—in either the R- or S-configuration. In some embodiments,R^(4*) and R^(2*) together designate the biradical —O—CH₂—O—CH₂— (Sethat al., 2010, J. Org. Chem).

In some embodiments, R^(4*) and R^(2*) together designate the biradical—O—NR—CH₃—(Seth at al., 2010, J. Org. Chem).

In some embodiments, the LNA units have a structure selected from thefollowing group:

In some embodiments, R^(1*), R², R³, R⁵, R^(5*) are independentlyselected from the group consisting of hydrogen, halogen, C₁₋₆ alkyl,substituted C₁₋₆ alkyl, C₂₋₆ alkenyl, substituted C₂₋₆ alkenyl, C₂₋₆alkynyl or substituted C₂₋₆ alkynyl, C₁₋₆ alkoxyl, substituted C₁₋₆alkoxyl, acyl, substituted acyl, C₁₋₆ aminoalkyl or substituted C₁₋₆aminoalkyl. For all chiral centers, asymmetric groups may be found ineither R or S orientation.

In some embodiments, R^(1*), R², R³, R⁵, R^(5*) are hydrogen.

In some embodiments, R^(1*), R², R³ are independently selected from thegroup consisting of hydrogen, halogen, C₁₋₆ alkyl, substituted C₁₋₆alkyl, C₂₋₆ alkenyl, substituted C₂₋₆ alkenyl, C₂₋₆ alkynyl orsubstituted C₂₋₆ alkynyl, C₁₋₆ alkoxyl, substituted C₁₋₆ alkoxyl, acyl,substituted acyl, C₁₋₆ aminoalkyl or substituted C₁₋₆ aminoalkyl. Forall chiral centers, asymmetric groups may be found in either R or Sorientation.

In some embodiments, R^(1*), R², R³ are hydrogen.

In some embodiments, R⁵ and R^(5*) are each independently selected fromthe group consisting of H, —CH₃, —CH₂—CH₃, —CH₂—O—CH₃, and —CH═CH₂.Suitably in some embodiments, either R⁵ or R^(5*) are hydrogen, where asthe other group (R⁵ or R^(5*) respectively) is selected from the groupconsisting of C₁₋₅ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, substituted C₁₋₆alkyl, substituted C₂₋₆ alkenyl, substituted C₂₋₆ alkynyl or substitutedacyl (—C(═O)—); wherein each substituted group is mono or polysubstituted with substituent groups independently selected from halogen,C₁₋₆ alkyl, substituted C₁₋₆ alkyl, C₂₋₆ alkenyl, substituted C₂₋₆alkenyl, C₂₋₆ alkynyl, substituted C₂₋₆ alkynyl, OJ₁, SJ₁, NJ₁J₂, N₃,COOJ₁, CN, O—C(═O)NJ₁J₂, N(H)C(═NH)NJ,J₂ or N(H)C(═X)N(H)J₂ wherein X isO or S; and each J₁ and J₂ is, independently, H, C₁₋₆ alkyl, substitutedC₁₋₆ alkyl, C₂₋₆ alkenyl, substituted C₂₋₆ alkenyl, C₂₋₆ alkynyl,substituted C₂₋₆ alkynyl, C₁₋₆ aminoalkyl, substituted C₁₋₆ aminoalkylor a protecting group. In some embodiments either R⁵ or R^(5*) issubstituted C₁₋₆ alkyl. In some embodiments either R⁵ or R^(5*) issubstituted methylene wherein preferred substituent groups include oneor more groups independently selected from F, NJ₁J₂, N₃, CN, OJ₁, SJ₁,O—C(═O)NJ₁J₂, N(H)C(═NH)NJ, J₂ or N(H)C(O)N(H)J₂. In some embodimentseach J₁ and J₂ is, independently H or C₁₋₆ alkyl. In some embodimentseither R⁵ or R^(5*) is methyl, ethyl or methoxymethyl. In someembodiments either R⁵ or R^(5*) is methyl. In a further embodimenteither R⁵ or R^(5*) is ethylenyl. In some embodiments either R⁵ orR^(5*) is substituted acyl. In some embodiments either R⁵ or R^(5*) isC(═O)NJ₁J₂. For all chiral centers, asymmetric groups may be found ineither R or S orientation. Such 5′ modified bicyclic nucleotides aredisclosed in WO 2007/134181, which is hereby incorporated by referencein its entirety.

In some embodiments B is a nucleobase, including nucleobase analoguesand naturally occurring nucleobases, such as a purine or pyrimidine, ora substituted purine or substituted pyrimidine, such as a nucleobasereferred to herein, such as a nucleobase selected from the groupconsisting of adenine, cytosine, thymine, adenine, uracil, and/or amodified or substituted nucleobase, such as 5-thiazolo-uracil,2-thio-uracil, 5-propynyluracil, 2′thio-thymine, 5-methyl cytosine,5-thiozolo-cytosine, 5-propynyl-cytosine, and 2,6-diaminopurine.

In some embodiments, R^(4*) and R^(2*) together designate a biradicalselected from —C(R^(a)R^(b))—O—, —C(R^(a)R^(b))—C(R^(c)R^(d))—O—,—C(R^(a)R^(b))—C(R^(c)R^(d))—C(R^(e)R^(f))O—,—C(R^(a)R^(b))—O—C(R^(c)R^(d))—, —C(R^(a)R^(b))—O—C(R^(c)R^(d))—O—,—C(R^(a)R^(b))—C(R^(c)R^(d))—,—C(R^(a)R^(b))—C(R^(c)R^(d))—C(R^(e)R^(f))—,C(R^(a))═C(R^(b))—C(R^(c)R^(d))—, —C(R^(a)R^(b))—N(R^(c))—,—C(R^(a)R^(b))—C(R^(c)R^(d))—N(R^(e))—, —C(R^(a)R^(b))—N(R^(c))—O—, and—C(R^(a)R^(b))—S—, —C(R^(a)R^(b))—C(R^(c)R^(d))—S—, wherein R^(a),R^(b), R^(c), R^(d), R^(e), and R^(f) each is independently selectedfrom hydrogen, optionally substituted C₁₋₁₂-alkyl, optionallysubstituted C₂₋₁₂-alkenyl, optionally substituted C₂₋₁₂-alkynyl,hydroxy, C₁₋₁₂-alkoxy, C₂₋₁₂-alkoxyalkyl, C₂₋₁₂-alkenyloxy, carboxy,C₁₋₁₂-alkoxycarbonyl, C₁₋₁₂-alkylcarbonyl, formyl, aryl,aryloxy-carbonyl, aryloxy, arylcarbonyl, heteroaryl,heteroaryloxy-carbonyl, heteroaryloxy, heteroarylcarbonyl, amino, mono-and di(C₁₋₆-alkyl)amino, carbamoyl, mono- anddi(C₁₋₆-alkyl)-amino-carbonyl, amino-C₁₋₆-alkyl-aminocarbonyl, mono- anddi(C₁₋₆-alkyl)aminocarbonyl, C₁₋₆-alkyl-carbonylamino, carbamido,C₁₋₆-alkanoyloxy, sulphono, C₁₋₆-alkylsulphonyloxy, nitro, azido,sulphanyl, C₁₋₆-alkylthio, halogen, DNA intercalators, photochemicallyactive groups, thermochemically active groups, chelating groups,reporter groups, and ligands, where aryl and heteroaryl may beoptionally substituted and where two geminal substituents R^(a) andR^(b) together may designate optionally substituted methylene (═CH₂).For all chiral centers, asymmetric groups may be found in either R or Sorientation.

In a further embodiment R^(4*) and R^(2*) together designate a biradical(bivalent group) selected from —CH₂—O—, —CH₂—S—, —CH₂—NH—, —CH₂—N(CH₃)—,—CH₂—CH₂—O—, —CH₂—CH(CH₃)—, —CH₂—CH₂—S—, —CH₂—CH₂—NH—, —CH₂—CH₂—CH₂—,—CH₂—CH₂—CH₂—O—, —CH₂—CH₂—CH(CH₃)—, —CH═CH—CH₂—, —CH₂—O—CH₂—O—,—CH₂—NH—O—, —CH₂—N(CH₃)—O—, —CH₂—O—CH₂—, —CH(CH₃)—O—, and—CH(CH₂—O—CH₃)—O—, and/or, —CH₂—CH₂—, and —CH═CH— For all chiralcenters, asymmetric groups may be found in either R or S orientation.

In some embodiments, R^(4*) and R^(2*) together designate the biradicalC(R^(a)R^(b))—N(R^(c))—O—, wherein R^(a) and R^(b) are independentlyselected from the group consisting of hydrogen, halogen, C₁₋₆ alkyl,substituted C₁₋₆ alkyl, C₂₋₆ alkenyl, substituted C₂₋₆ alkenyl, C₂₋₆alkynyl or substituted C₂₋₆ alkynyl, C₁₋₆ alkoxyl, substituted C₁₋₆alkoxyl, acyl, substituted acyl, C₁₋₆ aminoalkyl or substituted C₁₋₆aminoalkyl, such as hydrogen, and; wherein R^(c) is selected from thegroup consisting of hydrogen, halogen, C₁₋₆ alkyl, substituted C₁₋₆alkyl, C₂₋₆ alkenyl, substituted C₂₋₆ alkenyl, C₂₋₆ alkynyl orsubstituted C₂₋₆ alkynyl, C₁₋₆ alkoxyl, substituted C₁₋₆ alkoxyl, acyl,substituted acyl, C₁₋₆ aminoalkyl or substituted C₁₋₆ aminoalkyl, suchas hydrogen.

In some embodiments, R^(4*) and R^(2*) together designate the biradicalC(R^(a)R^(b))—O—C(R^(c)R^(d))—O—, wherein R^(a), R^(b), R^(e), and R^(d)are independently selected from the group consisting of hydrogen,halogen, C₁₋₆ alkyl, substituted C₁₋₆ alkyl, C₂₋₆ alkenyl, substitutedC₂₋₆ alkenyl, C₂₋₆ alkynyl or substituted C₂₋₆ alkynyl, C₁₋₆ alkoxyl,substituted C₁₋₆ alkoxyl, acyl, substituted acyl, C₁₋₆ aminoalkyl orsubstituted C₁₋₆ aminoalkyl, such as hydrogen.

In some embodiments, R^(4*) and R^(2*) form the biradical —CH(Z)—O—,wherein Z is selected from the group consisting of C₁₋₆ alkyl, C₂₋₆alkenyl, C₂₋₆ alkynyl, substituted C₁₋₆ alkyl, substituted C₂₋₆ alkenyl,substituted C₂₋₆ alkynyl, acyl, substituted acyl, substituted amide,thiol or substituted thio; and wherein each of the substituted groups,is, independently, mono or poly substituted with optionally protectedsubstituent groups independently selected from halogen, oxo, hydroxyl,OJ₁, NJ₁J₂, SJ₁, N₃, OC(═X)J₁, OC(═X)NJ₁J₂, NJ³C(═X)NJ₁J₂ and CN,wherein each J₁, J₂ and J₃ is, independently, H or C₁₋₆ alkyl, and X isO, S or NJ₁. In some embodiments Z is C₁₋₆ alkyl or substituted C₁₋₆alkyl. In some embodiments Z is methyl. In some embodiments Z issubstituted C₁₋₆ alkyl. In some embodiments said substituent group isC₁₋₆ alkoxy. In some embodiments Z is CH₃OCH₂—. For all chiral centers,asymmetric groups may be found in either R or S orientation. Suchbicyclic nucleotides are disclosed in U.S. Pat. No. 7,399,845 which ishereby incorporated by reference in its entirety. In some embodiments,R^(1*), R², R³, R⁵, R^(5*) are hydrogen. In some some embodiments,R^(1*), R², R^(3*) are hydrogen, and one or both of R⁵, R^(5*) may beother than hydrogen as referred to above and in WO 2007/134181.

In some embodiments, R^(4*) and R^(2*) together designate a biradicalwhich comprise a substituted amino group in the bridge such as consistor comprise of the biradical —CH₂—) N(R^(c))—, wherein R^(c) is C₁₋₁₂alkyloxy. In some embodiments R^(4*) and R^(2*) together designate abiradical —Cq₃q₄-NOR—, wherein q₃ and q₄ are independently selected fromthe group consisting of hydrogen, halogen, C₁₋₆ alkyl, substituted C₁₋₆alkyl, C₂₋₆ alkenyl, substituted C₂₋₆ alkenyl, C₂₋₆ alkynyl orsubstituted C₂₋₆ alkynyl, C₁₋₆ alkoxyl, substituted C₁₋₆ alkoxyl, acyl,substituted acyl, C₁₋₆ aminoalkyl or substituted C₁₋₆ aminoalkyl;wherein each substituted group is, independently, mono or polysubstituted with substituent groups independently selected from halogen,OJ₁, SJ₁, NJ₁J₂, COOJ₁, CN, O—C(═O)NJ₁J₂, N(H)C(═NH)N J₁J₂ orN(H)C(═X═N(H)J₂ wherein X is O or S; and each of J₁ and J₂ is,independently, H, C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₁₋₆aminoalkyl or a protecting group. For all chiral centers, asymmetricgroups may be found in either R or S orientation. Such bicyclicnucleotides are disclosed in WO2008/150729 which is hereby incorporatedby reference in its entirety. In some embodiments, R^(1*), R², R³, R⁵,R^(5*) are independently selected from the group consisting of hydrogen,halogen, C₁₋₆ alkyl, substituted C₁₋₆ alkyl, C₂₋₆ alkenyl, substitutedC₂₋₆ alkenyl, C₂₋₆ alkynyl or substituted C₂₋₆ alkynyl, C₁₋₆ alkoxyl,substituted C₁₋₆ alkoxyl, acyl, substituted acyl, C₁₋₆ aminoalkyl orsubstituted C₁₋₆ aminoalkyl. In some embodiments, R^(1*), R², R³, R⁵,R^(5*) are hydrogen. In some embodiments, R^(1*), R², R³ are hydrogenand one or both of R⁵, R^(5*) may be other than hydrogen as referred toabove and in WO 2007/134181. In some embodiments R^(4*) and R^(2*)together designate a biradical (bivalent group) C(R^(a)R^(b))—O—,wherein R^(a) and R^(b) are each independently halogen, C₁-C₁₂ alkyl,substituted C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, substituted C₂-C₁₂ alkenyl,C₂-C₁₂ alkynyl, substituted C₂-C₁₂ alkynyl, C₁-C₁₂ alkoxy, substitutedC₁-C₁₂ alkoxy, OJ₁ SJ₁, SOJ₁, SO₂J₁, NJ₁J₂, N₃, CN, C(═O)OJ₁,C(═O)NJ₁J₂, C(═O)J₁, O—C(═O)NJ₁J₂, N(H)C(═NH)NJ₁J₂, N(H)C(═O)NJ₁J₂ orN(H)C(═S)NJ₁J₂; or R^(a) and R^(b) together are ═C(q3)(q4); q₃ and q₄are each, independently, H, halogen, C₁-C₁₂alkyl or substituted C₁-C₁₂alkyl; each substituted group is, independently, mono or polysubstituted with substituent groups independently selected from halogen,C₁-C₆ alkyl, substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, substituted C₂-C₆alkenyl, C₂-C₆ alkynyl, substituted C₂-C₆ alkynyl, OJ₁, SJ₁, NJ₁J₂, N₃,CN, C(═O)OJ₁, C(═O)NJ₁J₂, C(═O)J₁, O—C(═O)NJ₁J₂, N(H)C(═O)NJ₁J₂ orN(H)C(═S)NJ₁J₂ and; each J₁ and J₂ is, independently, H, C1-C₆ alkyl,substituted C1-C₆ alkyl, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆alkynyl, substituted C₂-C₆ alkynyl, C1-C₆ aminoalkyl, substituted C1-C₆aminoalkyl or a protecting group. Such compounds are disclosed inWO2009006478A, hereby incorporated in its entirety by reference.

In some embodiments, R^(4*) and R^(2*) form the biradical-Q-, wherein Qis C(q₁)(q₂)C(q₃)(q₄), C(q₁)═C(q₃), C[═C(q₁)(q₂)]-C(q₃)(q₄) orC(q₁)(q₂)-C[═C(q₃)(q₄)]; q₁, q₂, q₃, q₄ are each independently. H,halogen, C₁₋₁₂ alkyl, substituted C₁₋₁₂ alkyl, C₂₋₁₂ alkenyl,substituted C₁₋₁₂ alkoxy, OJ₁, SJ₁, SOJ₁, SO₂J₁, NJ₁J₂, N₃, CN,C(═O)OJ₁, C(═O)—NJ₁J₂, C(═O) J₁, —C(═O)NJ₁J₂, N(H)C(═NH)NJ₁J₂,N(H)C(═O)NJ₁J₂ or N(H)C(═S)NJ₁J₂; each J₁ and J₂ is, independently, H,C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₁₋₆ aminoalkyl or a protectinggroup; and, optionally wherein when Q is C(q₁)(q₂)(q₃)(q₄) and one of q₃or q₄ is CH₃ then at least one of the other of q₃ or q₄ or one of q₁ andq₂ is other than H. In some embodiments, R^(1*), R², R³, R⁵, R^(5*) arehydrogen. For all chiral centers, asymmetric groups may be found ineither R or S orientation. Such bicyclic nucleotides are disclosed inWO2008/154401 which is hereby incorporated by reference in its entirety.In some embodiments, R^(1*), R², R³, R⁵, R^(5*) are independentlyselected from the group consisting of hydrogen, halogen, C₁₋₆ alkyl,substituted C₁₋₆ alkyl, C₂₋₆ alkenyl, substituted C₂₋₆ alkenyl, C₂₋₆alkynyl or substituted C₂₋₆ alkynyl, C₁₋₆ alkoxyl, substituted C₁₋₆alkoxyl, acyl, substituted acyl, C₁₋₆ aminoalkyl or substituted C₁₋₆aminoalkyl. In some embodiments, R^(1*), R², R³, R⁵, R^(5*) arehydrogen. In some embodiments, R^(1*), R², R³ are hydrogen and one orboth of R⁵, R^(5*) may be other than hydrogen as referred to above andin WO 2007/134181 or WO2009/067647 (alpha-L-bicyclic nucleic acidsanalogs).

Further bicyclic nucleoside analogues and their use in antisenseoligonucleotides are disclosed in WO2011/115818, WO2011/085102,WO2011/017521, WO09/100320, WO10/036698, WO09/124295 & WO09/006478. Suchnucleoside analogues may in some aspects be useful in the compounds ofpresent invention.

In some embodiments the LNA used in the oligonucleotide compounds of theinvention preferably has the structure of the general formula II:

wherein Y is selected from the group consisting of —O—, —CH₂O—, —S—,—NH—, N(R^(e)) and/or —CH₂—; Z and Z* are independently selected amongan internucleotide linkage, R^(H), a terminal group or a protectinggroup; B constitutes a natural or non-natural nucleotide base moiety(nucleobase), and R^(H) is selected from hydrogen and C₁₋₄-alkyl; R^(a),R^(b) R^(c), R^(d) and R^(e) are, optionally independently, selectedfrom the group consisting of hydrogen, optionally substitutedC₁₋₁₂-alkyl, optionally substituted C₂₋₁₂-alkenyl, optionallysubstituted C₂₋₁₂-alkynyl, hydroxy, C₁₋₁₂-alkoxy, C₂₋₁₂-alkoxyalkyl,C₂₋₁₂-alkenyloxy, carboxy, C₁₋₁₂-alkoxycarbonyl, C₁₋₁₂-alkylcarbonyl,formyl, aryl, aryloxy-carbonyl, aryloxy, arylcarbonyl, heteroaryl,heteroaryloxy-carbonyl, heteroaryloxy, heteroarylcarbonyl, amino, mono-and di(C₁₋₆-alkyl)amino, carbamoyl, mono- anddi(C₁₋₆-alkyl)-amino-carbonyl, amino-C₁₋₆-alkyl-aminocarbonyl, mono- anddi(C₁₋₆-alkyl)amino-C₁₋₆-alkyl-aminocarbonyl, C₁₋₆-alkyl-carbonylamino,carbamido, C₁₋₆-alkanoyloxy, sulphono, C₁₋₆-alkylsulphonyloxy, nitro,azido, sulphanyl, C₁₋₆-alkylthio, halogen, DNA intercalators,photochemically active groups, thermochemically active groups, chelatinggroups, reporter groups, and ligands, where aryl and heteroaryl may beoptionally substituted and where two geminal substituents R^(a) andR^(b) together may designate optionally substituted methylene (═CH₂);and R^(H) is selected from hydrogen and C₁₋₄-alkyl. In some embodimentsR^(a), R^(b) R^(c), R^(d) and R^(e) are, optionally independently,selected from the group consisting of hydrogen and C₁₋₆ alkyl, such asmethyl. For all chiral centers, asymmetric groups may be found in eitherR or S orientation, for example, two exemplary stereochemical isomersinclude the beta-D and alpha-L isoforms, which may be illustrated asfollows:

Specific exemplary LNA units are shown below:

The term “thio-LNA” comprises a locked nucleotide in which Y in thegeneral formula above is selected from S or —CH₂—S—. Thio-LNA can be inboth beta-D and alpha-L-configuration.

The term “amino-LNA” comprises a locked nucleotide in which Y in thegeneral formula above is selected from —N(H)—, N(R)—, CH₂—N(H)—, and—CH₂—N(R)— where R is selected from hydrogen and C₁₋₄-alkyl. Amino-LNAcan be in both beta-D and alpha-L-configuration.

The term “oxy-LNA” comprises a locked nucleotide in which Y in thegeneral formula above represents —O—. Oxy-LNA can be in both beta-D andalpha-L-configuration.

The term “ENA” comprises a locked nucleotide in which Y in the generalformula above is —CH₂—O— (where the oxygen atom of —CH₂—O— is attachedto the 2′-position relative to the base B). R^(e) is hydrogen or methyl.

In some exemplary embodiments LNA is selected from beta-D-oxy-LNA,alpha-L-oxy-LNA, beta-D-amino-LNA and beta-D-thio-LNA, in particularbeta-D-oxy-LNA.

LNA-modified antisense oligonucleotides may be used in combinations. Forinstance, a cocktail of several different LNA modified oligonucleotides,directed against different regions of the same gene, may be administeredsimultaneously or separately.

Headmer

A “headmen” is defined as an oligomer that comprises a region X′ and aregion Y′ that is contiguous thereto, with the 5′-most unit of region Y′linked to the 3′-most unit of region X′. Region X′ comprises acontiguous stretch of non-RNase recruiting nucleoside analogues andregion Y′ comprises a contiguous stretch (such as at least 7 contiguousunits) of DNA units or nucleoside analogue units recognizable andcleavable by the RNase.

Tailmer

A “tailmer” is defined as an oligomer that comprises a region X″ and aregion Y″ that is contiguous thereto, with the 5′-most unit of region Y″linked to the 3′-most unit of the region X″. Region X″ comprises acontiguous stretch (such as at least 7 contiguous units) of DNA units ornucleoside analogue units recognizable and cleavable by the RNase, andregion X″ comprises a contiguous stretch of non-RNase recruitingnucleoside analogues.

Chimeric Oligomers/Mixmers

“Chimeric” oligomers, called “mixmers”, consist of an alternatingcomposition of (i) DNA units or nucleoside analogue units recognizableand cleavable by RNase, and (ii) non-RNase recruiting nucleosideanalogue units.

Photochemically Active Groups

In the present context, the term “photochemically active groups” refersto compounds which are able to undergo chemical reactions uponirradiation with light. Illustrative examples of functional groupsherein are quinones, especially 6-methyl-1,4-naphtoquinone,anthraquinone, naphtoquinone, and 1,4-dimethyl-anthraquinone,diazirines, aromatic azides, benzophenones, psoralens, diazo compounds,and diazirino compounds.

Based On

As used herein, the term “based on” means that the oligomer or theoligomer of the oligomer conjugate comprises at least 80%, preferably atleast 85%, preferably at least 90%, preferably at least 95%, preferablyall of the nucleotides of the core motif or sequence and optionallywherein one or more of the nucleotides may be a modified nucleotide.

Accordingly, for certain embodiments, the term “based on” means that theoligomer or the oligomer of the oligomer conjugate comprises all of thenucleotides of the core motif or sequence and optionally wherein one ormore of the nucleotides may be a modified nucleotide.

For certain embodiments, the term “based on” means that the oligomer orthe oligomer of the oligomer conjugate comprises all of the nucleotidesof the core motif or sequence and wherein one or more of the nucleotidesis a modified nucleotide.

For certain embodiments, the term “based on” means that the oligomer orthe oligomer of the oligomer conjugate comprises at least 80%,preferably at least 85%, preferably at least 90%, preferably at least95%, preferably all of the nucleotides of the core motif or sequence andwherein one or more of the nucleotides may be a modified nucleotide; andwherein said oligomer is a gapmer of the motif X-Y-Z wherein each of Xand Z is independently a wing comprising at least one modifiednucleotide and Y is a central region of nucleotides.

Accordingly, for certain embodiments, the term “based on” means that theoligomer or the oligomer of the oligomer conjugate comprises all of thenucleotides of the core motif or sequence and wherein one or more of thenucleotides may be a modified nucleotide; and wherein said oligomer is agapmer of the motif X-Y-Z wherein each of X and Z is independently awing comprising at least one modified nucleotide and Y is a centralregion of nucleotides.

Stability

The term “stability” in reference to duplex or triplex formationgenerally designates how tightly an antisense oligonucleotide binds toits intended target sequence; more particularly, “stability” designatesthe free energy of formation of the duplex or triplex underphysiological conditions. Melting temperature under a standard set ofconditions, e.g., as described below, is a convenient measure of duplexand/or triplex stability. Preferably, antisense oligonucleotides of theinvention are selected that have melting temperatures of at least 45° C.when measured in 100 mM NaCl, 0.1 mM EDTA and 10 mM phosphate bufferaqueous solution, pH 7.0 at a strand concentration of both the antisenseoligonucleotide and the target nucleic acid of 1.5 μM. Thus, when usedunder physiological conditions, duplex or triplex formation will besubstantially favored over the state in which the antisenseoligonucleotide and its target are dissociated. It is understood that astable duplex or triplex may in some embodiments include mismatchesbetween base pairs and/or among base triplets in the case of triplexes.Preferably, LNA modified antisense oligonucleotides of the inventionform perfectly matched duplexes and/or triplexes with their targetnucleic acids.

Potent Inhibitor

In some embodiments the oligomer or oligomer conjugate is a potentinhibitor, in particular of HBx or HBsAg.

As used herein, the phrase “potent inhibitor” refers to an oligomer withan IC50 of less than 5 nM as determined by a lipofectamin transfectionassay. In some embodiments, the IC50 is less than 4 nM, such as lessthan 2 nM.

A “potent inhibitor” as determined by “gymnosis”, where gymnosisdescribes the delivery of the oligomer to the cultured cell without theuse of a transfection agent, refers to an IC50 of less than 5 μm in agymnosis assay or in a in vivo AAV/HBV Mouse Model. In some embodiments,the IC50 is less than 2 μm, such as less than 1 μm.

Treat/Treatment

The terms “treat”/“treatment” etc. include one or more of to cure, toalleviate, to prevent or to detect. In certain preferred embodiments,the terms “treat”/“treatment” etc. mean to cure or to alleviate. Theterm “alleviate” includes to alleviate the symptoms and/or theconditions attributed with or associated with a viral disorder.

Thus, in some aspects, the present invention relates to oligomers oroligomer conjugates suitable for, and uses thereof and methods usingsame, to cure, to alleviate, to prevent or to detect a viral disorder.

Accordingly, in certain aspects, the present invention relates tooligomers or oligomer conjugates suitable for, and uses thereof andmethods using same, to cure or to alleviate a viral disorder.

In some embodiments, the term ‘treatment’ as used herein refers to bothtreatment of an existing disease (e.g. a disease or disorder as hereinreferred to), or prevention of a disease, i.e. prophylaxis. It willtherefore be recognised that treatment as referred to herein may, Insome embodiments, be prophylactic.

“Treating” a disease or condition in a subject or “treating” a subjecthaving a disease or condition refers to subjecting the individual to apharmaceutical treatment, e.g., the administration of a drug, such thatat least one symptom of the disease or condition is decreased orstabilized.

A patient who is in need of treatment is a patient suffering from orlikely to suffer from the disease or disorder.

By “treating prophyllactically” a disease or condition in a subject ismeant reducing or eliminating the risk of developing (i.e., theincidence) of or reducing the severity of the disease or condition priorto the appearance of at least one symptom of the disease.

Agent

The term “agent” means any compound, for example, an antibody, or atherapeutic agent, a detectable label (e.g., a marker, tracer, orimaging compound).

Therapeutic Agent

The term “therapeutic agent” means any compound having a biologicalactivity. Therapeutic agents may be useful for treating conditions ordiseases. Specific therapeutic agents according to the invention may beoligomers. Specific therapeutic agents may be oligomers or oligomerconjugates according to the present invention.

Delivering Said Oligomer to the Liver

As used herein, the term “delivering said oligomer to the liver” relatesto the process by which an oligomer is brought into contact with, orinto proximity to the cells or tissues of the liver. This includes thedelivery of the oligomer to the blood supply within or surrounding theliver. The oligomer may be delivered or carried from the site of entryinto the body to the liver or tissues surrounding the liver.

Delivering Said Oligomer to a Hepatocyte

As used herein, the term “delivering said oligomer to a hepatocyte”relates to the process by which an oligomer is brought into contactwith, or into proximity to, a hepatocyte of the liver. This includes thedelivery of the oligomer to the blood supply within or surrounding ahepatocyte. The oligomer may be delivered or carried from the site ofentry into the body to a hepatocyte.

Viral Disorder

The term “viral disorder” in the context of the present invention refersto any disorder or disease which is associated with viral infection.

Pharmaceutical Carriers/Pharmaceutically Acceptable Carriers

“Pharmaceutical carriers” or “pharmaceutically acceptable carriers” areto be distinguished from the “carrier component” of the invention asdescribed above. “Pharmaceutical carrier” and “carrier component” are tobe treated as mutually exclusive terms in the context of the presentinvention.

Administering/Administration

The terms “administering” and “administration” are intended to mean amode of delivery including, without limitation, intra-arterially,intra-nasally, intra-peritoneally, intravenously, intramuscularly,sub-cutaneously, transdermally or per os. A daily dosage can be dividedinto one, two or more doses in a suitable form to be administered atone, two or more times throughout a time period.

DETAILED DESCRIPTION OF THE INVENTION Introduction

The present invention relates to oligomers (also referred to asoligomeric molecules) and/or oligomer conjugates.

The oligomers and oligomer conjugates are useful for the treatment of aviral disorder.

In particular embodiments, the oligomers or oligomer conjugates of theinvention are capable of modulating a target sequence in HBV HBx orHBsAg.

The oligomers of the invention may be conjugated to a carrier component.

Preferably, the carrier component may be capable of delivering theoligomer to the liver of a subject to be treated.

The present invention therefore employs oligomers or oligomer conjugatesfor use in modulating the function of nucleic acid molecules encodingHBV HBx or HBsAg, such as the HBV nucleic acid molecule presented as SEQID no 1 or SEQ ID No 2, and naturally occurring variants of such nucleicacid molecules encoding HBV HBx or HBV HBsAg.

Oligomer Features

The oligomer or the oligomer of the oligomer conjugate may consist orcomprise of a contiguous nucleotide sequence of from 8-50, 8-30, 8-25,8-20, 8-18, 8-17, 8-16, 8-15, 8-14, 8-13, or 8-12 nucleotides in length,preferably 8-16 nucleotides in length, more preferably 10 to 20nucleotides in length.

For some embodiments, the oligomer or the oligomer of the oligomerconjugate may comprise or consist of a contiguous nucleotide sequence offrom 10-50, such as 10-30, 10-20, 10-18, 10-17, 10-16, 10-15, 10-14,10-13, or 10-12 nucleotides in length, preferably 10-20 nucleotides inlength, more preferably 10 to 18 nucleotides in length, most preferably10 to 16 nucleotides in length.

For some embodiments, the oligomer or the oligomer of the oligomerconjugate may comprise or consist of a contiguous nucleotide sequence offrom 12-50, such as 12-30, 12-20, 12-18, 12-17, 12-16, 12-15, 12-14 or12-13 nucleotides in length, preferably 12-16 nucleotides in length.

For some embodiments, the oligomer or the oligomer of the oligomerconjugate may comprise or consist of a contiguous nucleotide sequence of8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 nucleotides inlength.

For some embodiments, the oligomers or the oligomer component of theoligomer conjugates of the present invention may comprise or consist ofa contiguous nucleotide sequence of a total of 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30contiguous nucleotides in length.

In some embodiments, the oligomer or the oligomer of the oligomerconjugate comprises or consists of a contiguous nucleotide sequence of atotal of from 8-22, such as 8-20, such as 8-18, such as 8-17 or 8-16,contiguous nucleotides in length.

In some embodiments, the oligomer or the oligomer of the oligomerconjugate comprises or consists of a contiguous nucleotide sequence of atotal of 8, 9, 10, 11, 12, 13, 14, 15, or 16 contiguous nucleotides inlength.

In some embodiments, the oligomer or the oligomer of the oligomerconjugate comprises or consists of a contiguous nucleotide sequence of atotal of 15 or 16 contiguous nucleotides in length.

In some embodiments, the oligomer or the oligomer of the oligomerconjugate according to the invention consists of no more than 22nucleotides, such as no more than 20 nucleotides, such as no more than18 nucleotides—such as 15, or 16 or 17 nucleotides.

In some embodiments, preferably the oligomer or the oligomer of theoligomer conjugate of the invention comprises less than 20 nucleotides.

It should be understood that when a range is given for an oligomer, orcontiguous nucleotide sequence length it includes the lower an upperlengths provided in the range, for example from (or between) 10-30,includes both 10 and 30.

The length of the oligonucleotide moieties is sufficiently large toensure that specific binding takes place only at the desired targetpolynucleotide and not at other fortuitous sites, as explained in manyreferences, e.g., Rosenberg et al., International applicationPCT/US92/05305; or Szostak et al., Meth. Enzymol, 68:419-429 (1979). Theupper range of the length is determined by several factors, includingthe inconvenience and expense of synthesizing and purifying oligomersgreater than about 30-40 nucleotides in length, the greater tolerance oflonger oligonucleotides for mismatches than shorter oligonucleotides,whether modifications to enhance binding or specificity are present,whether duplex or triplex binding is desired, and the like.

The oligomer or the oligomer of the oligomer conjugate may comprise orconsist of a simple sequence of natural occurring nucleotides—preferably2′-deoxynucleotides (referred to here generally as “DNA”), but alsopossibly ribonucleotides (referred to here generally as “RNA”), or acombination of such naturally occurring nucleotides and one or morenon-naturally occurring nucleotides, i.e. nucleotide analogues. Suchnucleotide analogues may suitably enhance the affinity of the oligomerfor the target sequence.

Examples of suitable and preferred nucleotide analogues are provided byWO2007/031091 or are referenced therein.

Examples of nucleotide analogues include nucleotides that have beenmodified. Examples of such modifications include modifying the sugarmoiety to provide a 2′-substituent group or to produce a bridged (lockednucleic acid) structure which enhances binding affinity and may alsoprovide increased nuclease resistance. These modified nucleotideanalogues can therefore be affinity-enhancing nucleotide analogues.

Incorporation of affinity-enhancing nucleotide analogues in the oligomeror the oligomer of the oligomer conjugate, such as LNA or 2′-substitutedsugars, can allow the size of the specifically binding oligomer to bereduced, and may also reduce the upper limit to the size of the oligomerbefore non-specific or aberrant binding takes place.

In some embodiments, the oligomer or the oligomer of the oligomerconjugate comprises at least 1 nucleotide analogue. In some embodimentsthe oligomer or the oligomer of the oligomer conjugate comprises atleast 2 nucleotide analogues. In some embodiments, the oligomer or theoligomer of the oligomer conjugate comprises from 2-8 nucleotideanalogues, e.g. 6 or 7 nucleotide analogues. In the by far mostpreferred embodiments, at least one of said nucleotide analogues is alocked nucleic acid (LNA); for example at least 2, 3 or at least 4, orat least 5, or at least 6, or at least 7, or 8, of the nucleotideanalogues may be LNA. In some embodiments all the nucleotides analoguesmay be LNA.

It will be recognised that when referring to a preferred nucleotidesequence motif or nucleotide sequence, which consists of onlynucleotides, the oligomers or the oligomers of the oligomer conjugatesof the invention which are defined by that sequence may comprise acorresponding nucleotide analogue in place of one or more of thenucleotides present in said sequence, such as LNA units or othernucleotide analogues, which raise the duplex stability/T, of theoligomer/target duplex (i.e. affinity enhancing nucleotide analogues).

A preferred nucleotide analogue is LNA, such as oxy-LNA (such asbeta-D-oxy-LNA, and alpha-L-oxy-LNA), and/or amino-LNA (such asbeta-D-amino-LNA and alpha-L-amino-LNA) and/or thio-LNA (such asbeta-D-thio-LNA and alpha-L-thio-LNA) and/or ENA (such as beta-D-ENA andalpha-L-ENA). Most preferred is beta-D-oxy-LNA.

In some embodiments the nucleotide analogues present within the oligomeror the oligomer of the oligomer conjugate of the invention (such as inregion W and region Y for gapmers—as mentioned herein) are independentlyselected from, for example: 2′-O-alkyl-RNA units, 2′-amino-DNA units,2′-fluoro-DNA units, LNA units, arabino nucleic acid (ANA) units,2′-fluoro-ANA units, HNA units, INA (intercalating nucleicacid—Christensen, 2002. Nucl. Acids. Res. 2002 30: 4918-4925, herebyincorporated by reference) units and 2′MOE units. In some embodimentsthere is only one of the above types of nucleotide analogues present inthe oligomer of the invention, or contiguous nucleotide sequencethereof.

In some embodiments the nucleotide analogues are 2′-O-methoxyethyl-RNA(2′MOE), 2′-fluoro-DNA units or LNA nucleotide analogues, and as suchthe oligonucleotide of the invention may comprise nucleotide analogueswhich are independently selected from these three types of analogue, ormay comprise only one type of analogue selected from the three types. Insome embodiments at least one of said nucleotide analogues is2′-MOE-RNA, such as 2, 3, 4, 5, 6, 7, 8, 9 or 10 2′-MOE-RNA nucleotideunits. In some embodiments at least one of said nucleotide analogues is2′-fluoro DNA, such as 2, 3, 4, 5, 6, 7, 8, 9 or 10 2′-fluoro-DNAnucleotide units.

In some embodiments, the oligomer or the oligomer of the oligomerconjugate according to the invention comprises at least one LockedNucleic Acid (LNA) unit, such as 1, 2, 3, 4, 5, 6, 7, or 8 LNA units,such as from 3-7 or 4 to 8 LNA units, or 3, 4, 5, 6 or 7 LNA units. Insome embodiments, all the nucleotide analogues are LNA.

In some embodiments, the oligomer or the oligomer of the oligomerconjugate may comprise both beta-D-oxy-LNA, and one or more of thefollowing LNA units: thio-LNA, amino-LNA, oxy-LNA, and/or ENA in eitherthe beta-D or alpha-L configurations or combinations thereof. In someembodiments all LNA cytosine units are 5′methyl-Cytosine.

In some embodiments of the invention, the oligomer or the oligomer ofthe oligomer conjugate may comprise both LNA and DNA units. Preferablythe combined total of LNA and DNA units is 10-25, such as 10-24,preferably 10-20, such as 10-18, even more preferably 12-16. In someembodiments of the invention, the nucleotide sequence of the oligomer,such as the contiguous nucleotide sequence consists of at least one LNAand the remaining nucleotide units are DNA units.

In some embodiments the oligomer or the oligomer of the oligomerconjugate comprises only LNA nucleotide analogues and naturallyoccurring nucleotides (such as RNA or DNA, most preferably DNAnucleotides), optionally with modified internucleotide linkages such asphosphorothioate.

In some embodiments, any mismatches between the nucleotide sequence ofthe oligomer and the target sequence are preferably found in regionsoutside the affinity enhancing nucleotide analogues. Examples of suchregions outside the affinity enhancing nucleotide analogues in gapmersinclude region X as referred to herein and/or region V as referred toherein and/or region Z as referred to herein, and/or at the site of nonmodified nucleotides in the oligonucleotide, and/or in regions which are5′ or 3′ to the contiguous nucleotide sequence.

In some embodiments, the oligomer or the oligomer of the oligomerconjugate of the invention does not comprise RNA (units). It ispreferred that the oligomer or the oligomer of the oligomer conjugateaccording to the invention is a linear molecule or is synthesised as alinear molecule. Preferably the oligomer or the oligomer of the oligomerconjugate is a single stranded molecule, and preferably does notcomprise short regions of, for example, at least 3, 4 or 5 contiguousnucleotides, which are complementary to equivalent regions within thesame oligomer (i.e. duplexes). In this regard, the oligomer or theoligomer of the oligomer conjugate is not (essentially) double stranded.In some embodiments, the oligomer or the oligomer of the oligomerconjugate is essentially single stranded. In various embodiments, theoligomer or the oligomer of the oligomer conjugate of the invention mayconsist entirely of the contiguous nucleotide region. Thus, preferablythe oligomer or the oligomer of the oligomer conjugate is notsubstantially self-complementary.

Hence, in certain aspects, the invention provides an oligomer or anoligomer conjugate having an oligomer component from 5-50, such as 5-30,or such as 5-20, such as 8-30, such as 8-20, such as 8-18, such as 8-16,such as 10-16, such as 10-15, such as 12-16, such as 12-15 nucleotidesin length which comprises a contiguous nucleotide sequence (a firstregion) of a total of at least 5 such as at least 8 nucleotides, whereinsaid contiguous nucleotide sequence (a first region) is at least 80%(e.g., 85%, 90%, 95%, 98%, or 99%) homologous to a region correspondingto the reverse complement of a HBV HBx or HBsAg gene or mRNA, such anysequence within any of SEQ ID No. 1, SEQ ID No. 2 and SEQ ID No. 3 ornaturally occurring variant thereof. Thus, for example, the oligomer orthe oligomer component of the oligomer conjugate hybridizes to a singlestranded nucleic acid molecule having the sequence of a portion of anyof SEQ ID No. 1, SEQ ID No. 2 and SEQ ID No. 3, preferably within any ofSEQ ID No. 1 and SEQ ID No. 2. The oligomer or oligomer component mayhybridize to a single stranded nucleic acid molecule having the sequenceof a portion sequence shown as position 200 to 1900 of SEQ ID No. 3).The oligomer or the oligomer component of the oligomer conjugate mayhybridize to a single stranded nucleic acid molecule having the sequenceshown as position 1264 to 1598 or 691 to 706 of SEQ ID NO: 3. Theoligomer or the oligomer component of the oligomer conjugate mayhybridize to a single stranded nucleic acid molecule with a sequenceselected from the group consisting of the following positions in SEQ IDNO 3: position 1 to 1944, position 157 to 1840, position 1196 to 1941,position 1376 to 1840 and position 3158-3182. Preferably, the oligomerconjugate hybridize to a single stranded nucleic acid molecule with asequence selected within position 1530 to 1598 of SEQ ID NO: 3, morepreferable within position 1577 to 1598 of SEQ ID NO: 3 and mostpreferably within position 1530 to 1543 of SEQ ID NO: 3. An oligomer orthe oligomer component of the oligomer conjugate may hybridize to asingle stranded nucleic acid molecule selected from the group consistingof positions: 1264-1278; 1265-1277; 1530-1543; 1530-1544; 1531-1543;1551-1565; 1551-1566; 1577-1589; 1577-1591; 1577-1592; 1578-1590;1578-1592; 1583-1598; 1584-1598; and 1585-1598; or 670-706, 691-705;691-706; 692-706; 693-706; and 694-706 of SEQ ID NO: 3. Preferably, anoligomer or the oligomer component of the oligomer conjugate mayhybridize to a single stranded nucleic acid molecule within position1530 to 1598 of SEQ ID NO: 3, more preferably within positions 1530-1543or positions 1577-1598 of SEQ ID NO: 3.

For certain embodiments, the invention provides an oligomer or anoligomer conjugate having an oligomer based on a core motif selectedfrom the group consisting of any one or more of:

(SEQ ID NO: 13) GCGTAAAGAGAGG; (SEQ ID NO: 11) GCGTAAAGAGAGGT;(SEQ ID NO: 20) AGCGAAGTGCACACG; (SEQ ID NO: 26) AGGTGAAGCGAAGTG;(SEQ ID NO 18) AGCGAAGTGCACACGG; (SEQ ID NO: 7) CGAACCACTGAACA;(SEQ ID NO 4) GAACCACTGAACAAA; (SEQ ID NO 5) CGAACCACTGAACAAA;(SEQ ID NO 6) CGAACCACTGAACAA; (SEQ ID NO 8) CGAACCACTGAAC (SEQ ID NO 9)CCGCAGTATGGATCG (SEQ ID NO: 10) CGCAGTATGGATC; (SEQ ID NO 12)CGCGTAAAGAGAGGT; (SEQ ID NO 14) AGAAGGCACAGACGG; (SEQ ID NO 15)GAGAAGGCACAGACGG (SEQ ID NO 16) GAAGTGCACACGG; (SEQ ID NO 17)GCGAAGTGCACACGG; (SEQ ID NO 19) CGAAGTGCACACG; (SEQ ID NO 27)AGGTGAAGCGAAGT; and (SEQ ID NO: 852) TAGTAAACTGAGCCA,which is capable of modulating a target sequence in HBx or HBsAg of HBVto treat a viral disorder.

The oligomer or the oligomer component of the oligomer conjugate may bebased on a sequence selected from the group consisting of any one ormore of:

(SEQ ID NO: 303) GCGtaaagagaGG; (SEQ ID NO: 301) GCGtaaagagaGGT;(SEQ ID NO: 618) GCGtaaagagAGG; (SEQ ID NO: 310) AGCgaagtgcacACG(SEQ ID NO: 668) AGgtgaagcgaAGTG; (SEQ ID NO: 308) AGCgaagtgcacaCGG;(SEQ ID NO: 297) CGAaccactgaACA; (SEQ ID NO: 300) CGCagtatggaTC;(SEQ ID NO: 315) AGGtgaagcgaagTGC; (SEQ ID NO: 316) AGGtgaagcgaaGTG;(SEQ ID NO: 294) GAAccactgaacAAA; (SEQ ID NO: 295) CGAaccactgaacAAA;(SEQ ID NO: 296) CGAaccactgaaCAA; (SEQ ID NO: 298) CGAaccactgaAC;(SEQ ID NO: 299) CCGcagtatggaTCG; (SEQ ID NO: 302) CGCgtaaagagaGGT;(SEQ ID NO: 304) AGAaggcacagaCGG; (SEQ ID NO: 305) GAGaaggcacagaCGG;(SEQ ID NO: 306) GAAgtgcacacGG; (SEQ ID NO: 307) GCGaagtgcacaCGG;(SEQ ID NO: 309) CGAagtgcacaCG; (SEQ ID NO: 585) GAAccactgaaCAAA;(SEQ ID NO: 588) CGAAccactgaacAAA (SEQ ID NO: 628) GAAgtgcacaCGG;(SEQ ID NO: 678) TAGtaaactgagCCA; (SEQ ID NO: 600) CGAaccactgAAC;(SEQ ID NO: 317) AGGtgaagcgaAGT; and (SEQ ID NO: 597) CGAaccactgAACA,wherein uppercase letters denote LNA units and lower case letters denoteDNA units.

In certain embodiments, the oligomer, or additional oligomer, oroligomer component of the oligomer conjugate, or oligomer component ofthe additional oligomer conjugate may comprise a sequence based on asequence selected from a list of:

(SEQ ID NO: 13) GCGTAAAGAGAGG; (SEQ ID NO: 11) GCGTAAAGAGAGGT;(SEQ ID NO 12) CGCGTAAAGAGAGGT; (SEQ ID NO 14) AGAAGGCACAGACGG;(SEQ ID NO 15) GAGAAGGCACAGACGG; (SEQ ID NO 18) AGCGAAGTGCACACGG;(SEQ ID NO 16) GAAGTGCACACGG; (SEQ ID NO 17) GCGAAGTGCACACGG;(SEQ ID NO: 20) AGCGAAGTGCACACG; (SEQ ID NO 19) CGAAGTGCACACG;(SEQ ID NO: 26) AGGTGAAGCGAAGTG; and (SEQ ID NO 27) AGGTGAAGCGAAGT.

In another embodiment the motif sequence is selected from:

(SEQ ID NO: 13) GCGTAAAGAGAGG; (SEQ ID NO: 11) GCGTAAAGAGAGGT;(SEQ ID NO: 20) AGCGAAGTGCACACG; (SEQ ID NO: 26) AGGTGAAGCGAAGTG; and(SEQ ID NO 18) AGCGAAGTGCACACGG

In another embodiment the motif sequence is selected from GCGTAAAGAGAGG(SEQ ID NO: 13) GCGTAAAGAGAGGT (SEQ ID NO: 11) and CGCGTAAAGAGAGGT (SEQID NO 12).

In another embodiment the motif sequence is selected from

(SEQ ID NO: 20) AGCGAAGTGCACACG; (SEQ ID NO: 26) AGGTGAAGCGAAGTG;(SEQ ID NO 18) AGCGAAGTGCACACGG; (SEQ ID NO 16) GAAGTGCACACGG;(SEQ ID NO 17) GCGAAGTGCACACGG; (SEQ ID NO 19) CGAAGTGCACACG and(SEQ ID NO 27) AGGTGAAGCGAAGT.

In another embodiment the motif sequence is selected from CGAACCACTGAACA(SEQ ID NO: 7); GAACCACTGAACAAA (SEQ ID NO 4); CGAACCACTGAACAAA (SEQ IDNO 5); CGAACCACTGAACAA (SEQ ID NO 6); CGAACCACTGAAC (SEQ ID NO 8) andTAGTAAACTGAGCCA (SEQ ID NO: 852).

In another embodiment the motif sequence is selected from

(SEQ ID NO 9) CCGCAGTATGGATCG and (SEQ ID NO: 10) CGCAGTATGGATC.

In another embodiment the motif sequence is selected from

(SEQ ID NO 14) AGAAGGCACAGACGG and (SEQ ID NO 15) GAGAAGGCACAGACGG.

In certain embodiments, the oligomer, or additional oligomer, oroligomer component of the oligomer conjugate, or oligomer component ofthe additional oligomer conjugate may comprise or consist of a sequenceselected from the group presented below:

(SEQ ID NO: 303) GCGtaaagagaGG; (SEQ ID NO: 301) GCGtaaagagaGGT;(SEQ ID NO: 618) GCGtaaagagAGG; (SEQ ID NO: 310) AGCgaagtgcacACG(SEQ ID NO: 668) AGgtgaagcgaAGTG; (SEQ ID NO: 308) AGCgaagtgcacaCGG;(SEQ ID NO: 297) CGAaccactgaACA; (SEQ ID NO: 300) CGCagtatggaTC;(SEQ ID NO: 315) AGGtgaagcgaagTGC; (SEQ ID NO: 316) AGGtgaagcgaaGTG;(SEQ ID NO: 294) GAAccactgaacAAA; (SEQ ID NO: 295) CGAaccactgaacAAA;(SEQ ID NO: 296) CGAaccactgaaCAA; (SEQ ID NO: 298) CGAaccactgaAC;(SEQ ID NO: 299) CCGcagtatggaTCG; (SEQ ID NO: 302) CGCgtaaagagaGGT;(SEQ ID NO: 304) AGAaggcacagaCGG; (SEQ ID NO: 305) GAGaaggcacagaCGG;(SEQ ID NO: 306) GAAgtgcacacGG; (SEQ ID NO: 307) GCGaagtgcacaCGG;(SEQ ID NO: 309) CGAagtgcacaCG; (SEQ ID NO: 585) GAAccactgaaCAAA;(SEQ ID NO: 588) CGAAccactgaacAAA (SEQ ID NO: 628) GAAgtgcacaCGG;(SEQ ID NO: 678) TAGtaaactgagCCA; (SEQ ID NO: 600) CGAaccactgAAC;(SEQ ID NO: 317) AGGtgaagcgaAGT; and (SEQ ID NO: 597) CGAaccactgAACA.wherein uppercase letters denote affinity enhancing nucleotide analoguesand lower case letters denote DNA units.

In another embodiment the sequence is selected from:

(SEQ ID NO: 303) GCGtaaagagaGG; (SEQ ID NO: 301) GCGtaaagagaGGT;(SEQ ID NO: 618) GCGtaaagagAGG; (SEQ ID NO: 302) CGCgtaaagagaGGT;(SEQ ID NO: 304) AGAaggcacagaCGG; (SEQ ID NO: 305) GAGaaggcacagaCGG;(SEQ ID NO: 306) GAAgtgcacacGG; (SEQ ID NO: 307) GCGaagtgcacaCGG;(SEQ ID NO: 628) GAAgtgcacaCGG; (SEQ ID NO: 308) AGCgaagtgcacaCGG;(SEQ ID NO: 309) CGAagtgcacaCG; (SEQ ID NO: 310) AGCgaagtgcacACG(SEQ ID NO: 315) AGGtgaagcgaagTGC; (SEQ ID NO: 316) AGGtgaagcgaaGTG;(SEQ ID NO: 317) AGGtgaagcgaAGT; and (SEQ ID NO: 668) AGgtgaagcgaAGTG.

In one embodiment the sequence is selected from:

(SEQ ID NO: 303) GCGtaaagagaGG; (SEQ ID NO: 301) GCGtaaagagaGGT;(SEQ ID NO: 618) GCGtaaagagAGG; (SEQ ID NO: 310) AGCgaagtgcacACG(SEQ ID NO: 668) AGgtgaagcgaAGTG; and (SEQ ID NO: 308) AGCgaagtgcacaCGG.

In one embodiment the sequence is selected from GCGtaaagagaGG (SEQ IDNO: 303) GCGtaaagagaGGT(SEQ ID NO: 301); GCGtaaagagAGG (SEQ ID NO: 618)and CGCgtaaagagaGGT (SEQ ID NO: 302).

In another embodiment the sequence is selected from AGCgaagtgcacACG (SEQID NO: 310); AGGtgaagcgaagTGC (SEQ ID NO: 315); AGGtgaagcgaaGTG (SEQ IDNO: 316); GAAgtgcacaCGG (SEQ ID NO: 628); AGgtgaagcgaAGTG (SEQ ID NO:668); AGCgaagtgcacaCGG (SEQ ID NO: 308); GAAgtgcacacGG (SEQ ID NO: 306);GCGaagtgcacaCGG (SEQ ID NO: 307); CGAagtgcacaCG (SEQ ID NO: 309); andAGGtgaagcgaAGT (SEQ ID NO: 317).

In another embodiment the sequence is selected from CGAaccactgaACA (SEQID NO: 297); GAAccactgaacAAA (SEQ ID NO: 294); CGAaccactgaacAAA (SEQ IDNO: 295); CGAaccactgaaCAA (SEQ ID NO: 296); CGAaccactgaAC (SEQ ID NO:298); CGAaccactgAACA (SEQ ID NO: 597) and TAGtaaactgagCCA (SEQ ID NO:678).

In another embodiment the sequence is selected from CCGcagtatggaTCG (SEQID NO: 299) and CGCagtatggaTC (SEQ ID NO: 300).

In another embodiment the sequence is selected from AGAaggcacagaCGG (SEQID NO: 304) and GAGaaggcacagaCGG (SEQ ID NO: 305).

The oligomer conjugate may be based on a sequence selected from thegroup consisting of any one or more of:

(SEQ ID NO: 815) 5′-GN2-C6 ca

t_(s)a_(s)a_(s)a_(s)g_(s)a_(s)g_(s)a_(s)

-3′ (SEQ ID NO: 814) 5′-GN2-C6 ca

t_(s)a_(s)a_(s)a_(s)g_(s)a_(s)g_(s)a_(s)

-3′ (SEQ ID NO: 825) 5′-GN2-C6 ca

_(s)t_(s)a_(s)a_(s)a_(s)g_(s)a_(s)g_(s)

-3′ (SEQ ID NO: 808) 5′-GN2-C6 ca

g_(s)a_(s)a_(s)g_(s)t_(s)g_(s)c_(s)a_(s)c_(s)

-3′ (SEQ ID NO: 826) 5′-GN2-C6 ca

_(s)g_(s)t_(s)g_(s)a_(s)a_(s)g_(s) ^(m)c_(s)g_(s)a_(s)

-3′ (SEQ ID NO: 807) 5′-GN2-C6 ca

g_(s)a_(s)a_(s)g_(s)t_(s)g_(s)c_(s)a_(s)c_(s)a_(s)

-3′ (SEQ ID NO: 799) 5′-GN2-C6 ca

c_(s)c_(s)a_(s)c_(s)t_(s)g_(s)a_(s)a_(s)c_(s)

-3′ (SEQ ID NO: 800) 5′-GN2-C6 ca

a_(s)c_(s)c_(s)a_(s)c_(s)t_(s)g_(s)a_(s)a_(s)c_(s)

-3′ (SEQ ID NO: 801) 5′-GN2-C6 ca

a_(s)c_(s)c_(s)a_(s)c_(s)t_(s)g_(s)a_(s)a_(s)

-3′ (SEQ ID NO: 802) 5′-GN2-C6 ca

c_(s)a_(s)g_(s)t_(s)a_(s)t_(s)g_(s)g_(s)a_(s)

-3′ (SEQ ID NO: 803) 5′-GN2-C6 ca

g_(s)t_(s)a_(s)a_(s)a_(s)g_(s)a_(s)g_(s)a_(s)

-3′ (SEQ ID NO: 804) 5′-GN2-C6 caA

a_(s)g_(s)g_(s)c_(s)a_(s)c_(s)a_(s)g_(s)a_(s)

-3′ (SEQ ID NO: 805) 5′-GN2-C6 ca

a_(s)a_(s)g_(s)g_(s)c_(s)a_(s)c_(s)a_(s)g_(s)a_(s)

-3′ (SEQ ID NO: 806) 5′-GN2-C6 ca

a_(s)a_(s)g_(s)t_(s)g_(s)c_(s)a_(s)c_(s)a_(s)

-3′ (SEQ ID NO: 809) 5′-GN2-C6 ca

t_(s)g_(s)a_(s)a_(s)g_(s) ^(m)c_(s)g_(s)a_(s)a_(s)g_(s)

-3′ (SEQ ID NO: 810) 5′-GN2-C6 ca

t_(s)g_(s)a_(s)a_(s)g_(s) ^(m)c_(s)g_(s)a_(s)a_(s)

-3′ (SEQ ID NO: 811) 5′-GN2-C6 ca

a_(s)c_(s)c_(s)a_(s)c_(s)t_(s)g_(s)a_(s)

-3′ (SEQ ID NO: 812) 5′-GN2-C6 ca

a_(s)c_(s)c_(s)a_(s)c_(s)t_(s)g_(s)a_(s)

-3′ (SEQ ID NO: 813) 5′-GN2-C6 ca

a_(s)g_(s)t_(s)a_(s)t_(s)g_(s)g_(s)a_(s)

-3′ (SEQ ID NO: 816) 5′-GN2-C6 ca

g_(s)t_(s)g_(s)c_(s)a_(s)c_(s)a_(s) ^(m)c_(s)

-3′ (SEQ ID NO: 817) 5′-GN2-C6 ca

a_(s)g_(s)t_(s)g_(s)c_(s)a_(s)c_(s)a_(s)

-3′ (SEQ ID NO: 818) 5′-GN2-C6 ca

t_(s)gsa_(s)a_(s)g_(s) ^(m)c_(s)g_(s)a_(s)

-3′ (SEQ ID NO: 819) 5′-GN2-C6 ca

_(s)c_(s)c_(s)a_(s)c_(s)t_(s)g_(s)a_(s)a_(s)

-3′ (SEQ ID NO: 820) 5′-GN2-C6 ca

_(s)c_(s)c_(s)a_(s)c_(s)t_(s)g_(s)a_(s)a_(s)c_(s)

-3′ (SEQ ID NO: 821) 5′-GN2-C6 ca

_(s)g_(s)t_(s)g_(s)c_(s)a_(s)c_(s)a_(s)

-3′ (SEQ ID NO: 822) 5′-GN2-C6 ca

_(s)t_(s)a_(s)a_(s)a_(s)c_(s)t_(s)g_(s)a_(s)g_(s)

s3′ (SEQ ID NO: 823) 5′-GN2-C6 ca

_(s)a_(s)c_(s)c_(s)a_(s)c_(s)t_(s)g_(s)

-3′ (SEQ ID NO: 824) 5′-GN2-C6 ca

_(s)a_(s)c_(s)c_(s)a_(s)c_(s)t_(s)g_(s)

-3′wherein uppercase letters denote beta-D-oxy-LNA units; lowercase lettersdenote DNA units; the subscript “s” denotes a phosphorothioate linkage;superscript m denotes a a DNA or beta-D-oxy-LNA unit containing a5-methylcytosine base; GN2-C6 denotes a GalNAc2 carrier component with aC6 linker.

In another embodiment the oligomer conjugate is selected from:

(SEQ ID NO: 815) 5′-GN2-C6 ca

t_(s)a_(s)a_(s)a_(s)g_(s)a_(s)g_(s)a_(s)

-3′ (SEQ ID NO: 814) 5′-GN2-C6 ca

t_(s)a_(s)a_(s)a_(s)g_(s)a_(s)g_(s)a_(s)

-3′ (SEQ ID NO: 825) 5′-GN2-C6 ca

_(s)t_(s)a_(s)a_(s)a_(s)g_(s)a_(s)g_(s)

-3′ (SEQ ID NO: 803) 5′-GN2-C6 ca

g_(s)t_(s)a_(s)a_(s)a_(s)g_(s)a_(s)g_(s)a_(s)

-3′ (SEQ ID NO: 804) 5′-GN2-C6 ca

a_(s)g_(s)g_(s)c_(s)a_(s)c_(s)a_(s)g_(s)a_(s)

-3′ (SEQ ID NO: 805) 5′-GN2-C6 ca

a_(s)a_(s)g_(s)g_(s)c_(s)a_(s)c_(s)a_(s)g_(s)a_(s)

-3′ (SEQ ID NO: 816) 5′-GN2-C6 ca

g_(s)t_(s)g_(s)c_(s)a_(s)c_(s)a_(s) ^(m)c_(s)

-3′ (SEQ ID NO: 806) 5′-GN2-C6 caG

a_(s)a_(s)g_(s)t_(s)g_(s)c_(s)a_(s)c_(s)a_(s)

-3′ (SEQ ID NO: 821) 5′-GN2-C6 ca

_(s)g_(s)t_(s)g_(s)c_(s)a_(s)c_(s)a_(s)

-3′ (SEQ ID NO: 807) 5′-GN2-C6 ca

g_(s)a_(s)a_(s)g_(s)t_(s)g_(s)c_(s)a_(s)c_(s)a_(s)

-3′ (SEQ ID NO: 817) 5′-GN2-C6 ca

a_(s)g_(s)t_(s)g_(s)c_(s)a_(s)c_(s)a_(s)

-3′ (SEQ ID NO: 808) 5′-GN2-C6 ca

g_(s)a_(s)a_(s)g_(s)t_(s)g_(s)c_(s)a_(s)c_(s)

-3′ (SEQ ID NO: 809) 5′-GN2-C6 ca

t_(s)g_(s)a_(s)a_(s)g_(s) ^(m)c_(s)g_(s)a_(s)a_(s)g_(s)

-3′ (SEQ ID NO: 810) 5′-GN2-C6 ca

t_(s)g_(s)a_(s)a_(s)g_(s) ^(m)c_(s)g_(s)a_(s)a_(s)

-3′ (SEQ ID NO: 818) 5′-GN2-C6 ca

t_(s)g_(s)a_(s)a_(s)g_(s) ^(m)c_(s)g_(s)a_(s)

-3′ (SEQ ID NO: 826) 5′-GN2-C6 ca

A_(s)G_(s)g_(s)t_(s)g_(s)a_(s)a_(s)g_(s) ^(m)c_(s)g_(s)a_(s)

-3′

In another embodiment the oligomer conjugate is selected from:

(SEQ ID NO: 815) 5′-GN2-C6 ca

t_(s)a_(s)a_(s)a_(s)g_(s)a_(s)g_(s)a_(s)

-3′ (SEQ ID NO: 814) 5′-GN2-C6 ca

G_(s)t_(s)a_(s)a_(s)a_(s)g_(s)a_(s)g_(s)a_(s)

-3′ (SEQ ID NO: 825) 5′-GN2-C6 ca

_(s)t_(s)a_(s)a_(s)a_(s)g_(s)a_(s)g_(s)

-3′ (SEQ ID NO: 808) 5′-GN2-C6 ca

g_(s)a_(s)a_(s)g_(s)t_(s)g_(s)c_(s)a_(s)c_(s)

-3′ (SEQ ID NO: 826) 5′-GN2-C6 ca

_(s)g_(s)t_(s)g_(s)a_(s)a_(s)g_(s) ^(m)c_(s)g_(s)a_(s)

-3′ (SEQ ID NO: 807) 5′-GN2-C6 ca

g_(s)a_(s)a_(s)g_(s)t_(s)g_(s)c_(s)a_(s)c_(s)a^(s)

-3′

In another embodiment the oligomer conjugate is selected from:

(SEQ ID NO: 815) 5′-GN2-C6 ca

t_(s)a_(s)a_(s)a_(s)g_(s)a_(s)g_(s)a_(s)

-3′ (SEQ ID NO: 814) 5′-GN2-C6 ca

t_(s)a_(s)a_(s)a_(s)g_(s)a_(s)g_(s)a_(s)

-3′ (SEQ ID NO: 825) 5′-GN2-C6 ca

_(s)t_(s)a_(s)a_(s)a_(s)g_(s)a_(s)g_(s)

-3′ (SEQ ID NO: 803) 5′-GN2-C6 ca

g_(s)t_(s)a_(s)a_(s)a_(s)g_(s)a_(s)g_(s)a_(s)

-3′

In another embodiment the oligomer conjugate is selected from:

(SEQ ID NO: 808) 5′-GN2-C6 ca

g_(s)a_(s)a_(s)g_(s)t_(s)g_(s)c_(s)a_(s)c_(s)

-3′ (SEQ ID NO: 809) 5′-GN2-C6 ca

t_(s)g_(s)a_(s)a_(s)g_(s) ^(m)c_(s)g_(s)a_(s)a_(s)g_(s)

-3′ (SEQ ID NO: 810) 5′-GN2-C6 ca

t_(s)g_(s)a_(s)a_(s)g_(s) ^(m)c_(s)g_(s)a_(s)a_(s)

-3′ (SEQ ID NO: 818) 5′-GN2-C6 ca

t_(s)g_(s)a_(s)a_(s)g_(s) ^(m)c_(s)g_(s)a_(s)

-3′ (SEQ ID NO: 821) 5′-GN2-C6 ca

_(s)g_(s)t_(s)g_(s)c_(s)a_(s)c_(s)a_(s)

-3′ (SEQ ID NO: 826) 5′-GN2-C6 ca

_(s)g_(s)t_(s)g_(s)a_(s)a_(s)g_(s) ^(m)c_(s)g_(s)a_(s)

-3′ (SEQ ID NO: 807) 5′-GN2-C6 ca

g_(s)a_(s)a_(s)g_(s)t_(s)g_(s)c_(s)a_(s)c_(s)a_(s)

-3′ (SEQ ID NO: 817) 5′-GN2-C6 ca

a_(s)g_(s)t_(s)g_(s)c_(s)a_(s)c_(s)a_(s)

-3′ (SEQ ID NO: 806) 5′-GN2-C6 ca

a_(s)a_(s)g_(s)t_(s)g_(s)c_(s)a_(s)c_(s)a_(s)

-3′ (SEQ ID NO: 816) 5′-GN2-C6 ca

g_(s)t_(s)g_(s)c_(s)a_(s)c_(s)a_(s) ^(m)c_(s)

-3′

In another embodiment the oligomer conjugate is selected from:

(SEQ ID NO: 799) 5′-GN2-C6 ca

c_(s)c_(s)a_(s)c_(s)t_(s)g_(s)a_(s)a_(s)c_(s)

-3′ (SEQ ID NO: 800) 5′-GN2-C6 ca

a_(s)c_(s)c_(s)a_(s)c_(s)t_(s)g_(s)a_(s)a_(s)c_(s)

-3′ (SEQ ID NO: 801) 5′-GN2-C6 ca

a_(s)c_(s)c_(s)a_(s)c_(s)t_(s)g_(s)a_(s)a_(s)

-3′ (SEQ ID NO: 811) 5′-GN2-C6 ca

a_(s)c_(s)c_(s)a_(s)c_(s)t_(s)g_(s)a_(s)

-3′ (SEQ ID NO: 812) 5′-GN2-C6 ca

a_(s)c_(s)c_(s)a_(s)c_(s)t_(s)g_(s)a_(s)

-3′ (SEQ ID NO: 824) 5′-GN2-C6 ca

_(s)a_(s)c_(s)c_(s)a_(s)c_(s)t_(s)g_(s)

-3′ (SEQ ID NO: 822) 5′-GN2-C6 ca

_(s)t_(s)a_(s)a_(s)a_(s)c_(s)t_(s)g_(s)a_(s)g_(s)

_(s) 3′

In another embodiment the oligomer conjugate is selected from:

(SEQ ID NO: 802) 5′-GN2-C6 ca

c_(s)a_(s)g_(s)t_(s)a_(s)t_(s)g_(s)g_(s)a_(s)

-3′ (SEQ ID NO: 813) 5′-GN2-C6 ca

a_(s)g_(s)t_(s)a_(s)t_(s)g_(s)g_(s)a_(s)

-3′

In another embodiment the oligomer conjugate is selected from:

(SEQ ID NO: 804) 5′-GN2-C6 ca

a_(s)g_(s)g_(s)c_(s)a_(s)c_(s)a_(s)g_(s)a_(s)

-3′ (SEQ ID NO: 805) 5′-GN2-C6 ca

a_(s)a_(s)g_(s)g_(s)c_(s)a_(s)c_(s)a_(s)g_(s)a_(s)

-3′

In some embodiments the oligomer or oligomer conjugate according to theinvention consists of or comprises or is based on the sequence motifGCGTAAAGAGAGG (SEQ ID NO: 13), or a sub-sequence of thereof.

In some embodiments the oligomer or oligomer conjugate according to theinvention consists of or comprises or is based on the sequence motifAGCGAAGTGCACACG (SEQ ID NO: 20), or a sub-sequence of thereof.

In some embodiments the oligomer or oligomer conjugate according to theinvention consists of or comprises or is based on the sequence motifGCGTAAAGAGAGGT (SEQ ID NO: 11), or a sub-sequence of thereof.

In some embodiments the oligomer or oligomer conjugate according to theinvention consists of or comprises or is based on the sequence motifAGGTGAAGCGAAGTG (SEQ ID NO: 26), or a sub-sequence of thereof.

In some embodiments the oligomer or oligomer conjugate according to theinvention consists of or comprises or is based on the sequence motifAGCGAAGTGCACACGG (SEQ ID NO 18), or a sub-sequence of thereof.

In some embodiments the oligomer or oligomer conjugate according to theinvention consists of or comprises or is based on the sequence motifCGAACCACTGAACA (SEQ ID NO: 7), or a sub-sequence of thereof.

In some embodiments the oligomer or oligomer conjugate according to theinvention consists of or comprises or is based on the sequence motifCCGCAGTATGGATCG (SEQ ID NO 9), or a sub-sequence of thereof.

In some embodiments the oligomer or oligomer conjugate according to theinvention consists of or comprises or is based on GCGtaaagagaGG (SEQ IDNO: 303).

In some embodiments the oligomer or oligomer conjugate according to theinvention consists of or comprises or is based on GCGtaaagagaGGT(SEQ IDNO: 301).

In some embodiments the oligomer or oligomer conjugate according to theinvention consists of or comprises or is based on GCGtaaagagAGG (SEQ IDNO: 618).

In some embodiments the oligomer or oligomer conjugate according to theinvention consists of or comprises or is based on AGCgaagtgcacACG (SEQID NO: 310).

In some embodiments the oligomer or oligomer conjugate according to theinvention consists of or comprises or is based on AGgtgaagcgaAGTG (SEQID NO: 668).

In some embodiments the oligomer or oligomer conjugate according to theinvention consists of or comprises or is based on AGCgaagtgcacaCGG (SEQID NO: 308).

In some embodiments the oligomer or oligomer conjugate according to theinvention consists of or comprises or is based on CGAaccactgaACA (SEQ IDNO: 297).

In some embodiments the oligomer or oligomer conjugate according to theinvention consists of or comprises or is based on CCGcagtatggaTCG (SEQID NO: 299)

In some embodiments the oligomer conjugate according to the inventionconsists of or comprises 5′-GN2-C6 caG_(s)^(m)C_(s)G_(s)t_(s)a_(s)a_(s)a_(s)g_(s)a_(s)g_(s)a_(s)G_(s)G-3′ (SEQ IDNO: 815).

In some embodiments the oligomer conjugate according to the inventionconsists of or comprises 5′-GN2-C6 caG_(s)^(m)C_(s)G_(s)t_(s)a_(s)a_(s)a_(s)g_(s)a_(s)g_(s)a_(s)G_(s)G_(s)T-3′(SEQ ID NO: 814).

In some embodiments the oligomer conjugate according to the inventionconsists of or comprises 5′-GN2-C6 caG_(s)^(m)C_(s)G_(s)t_(s)a_(s)a_(s)a_(s)g_(s)a_(s)g_(s)A_(s)G_(s)G-3′ (SEQ IDNO: 825).

In some embodiments the oligomer conjugate according to the inventionconsists of or comprises 5′-GN2-C6 caA_(s)G_(s)^(m)C_(s)g_(s)a_(s)a_(s)g_(s)t_(s)g_(s)c_(s)a_(s)c_(s)A_(s)^(m)C_(s)G-3′ (SEQ ID NO: 808).

In some embodiments the oligomer conjugate according to the inventionconsists of or comprises 5′-GN2-C6caA_(s)G_(s)g_(s)t_(s)g_(s)a_(s)a_(s)g_(s)^(m)c_(s)g_(s)a_(s)A_(s)G_(s)T_(s)G-3′ (SEQ ID NO: 826).

In some embodiments the oligomer conjugate according to the inventionconsists of or comprises 5′-GN2-C6 caA_(s)G_(s)^(m)C_(s)g_(s)a_(s)a_(s)g_(s)t_(s)g_(s)c_(s)a_(s)c_(s)a_(s)^(m)C_(s)G_(s)G-3′ (SEQ ID NO: 807).

In some embodiments the oligomer conjugate according to the inventionconsists of or comprises 5′-GN2-C6ca^(m)C_(s)G_(s)A_(s)a_(s)c_(s)c_(s)a_(s)c_(s)t_(s)g_(s)a_(s)A_(s)^(m)C_(s)A-3′ (SEQ ID NO: 811).

In some embodiments the oligomer conjugate according to the inventionconsists of or comprises 5′-GN2-C6 ca^(m)C_(s)^(m)C_(s)G_(s)c_(s)a_(s)g_(s)t_(s)a_(s)t_(s)g_(s)g_(s)a_(s)T_(s)^(m)C_(s)G-3′ (SEQ ID NO: 802).

Gapmer

Preferably, the oligomer or the oligomer component of the oligomerconjugate of the invention is a gapmer (sometimes referred to as agapmer oligomer). Preferably, the oligomer region comprises a2′-deoxyribonucleotide gap region flanked on each side by a wing,wherein each wing independently comprises one or more LNA units.

Typically, a gapmer oligomer of the present invention or the gapmeroligomer component of the oligomer conjugate of the invention can berepresented by any one of the following formulae:W-X-Y;V-W-X-Y;W-X-Y-Z;V-W-X-Y-Z;

wherein for each formula:

-   -   W represents a region of one or more affinity enhancing        nucleotide analogues (region W)    -   X represents a region comprising a stretch of nucleotides        capable of recruiting an RNAse (region X)    -   Y represents a region of one or more affinity enhancing        nucleotide analogues (region Y)    -   V represents a region of one or more nucleotide units (region V)    -   Z represents a region of one or more nucleotide units (region        Z).

Any one of regions V, W, X, Y or Z may contain additional nucleotides oraffinity enhancing nucleotide analogues.

Typically, therefore, a gapmer oligomer is an oligomer which comprises acontiguous stretch of nucleotides which is capable of recruiting anRNAse, such as RNAseH, such as a region of at least 6 or 7 DNAnucleotides that is region X; wherein region X is flanked both 5′ and 3′by regions of affinity enhancing nucleotide analogues, such as from 1-6nucleotide analogues 5′ and 3′ to the contiguous stretch of nucleotideswhich is capable of recruiting RNAse—these regions are region W andregion Y respectively.

In some embodiments, the units which are capable of recruiting RNAse areselected from the group consisting of DNA units, alpha-L-LNA units, C4′alkylayted DNA units (see PCT/EP2009/050349 and Vester et al., Bioorg.Med. Chem. Lett. 18 (2008) 2296-2300, hereby incorporated by reference),and UNA (unlinked nucleic acid) nucleotides (see Fluiter et al., Mol.Biosyst., 2009, 10, 1039 hereby incorporated by reference). UNA isunlocked nucleic acid, typically where the C2-C3 C—C bond of the ribosehas been removed, forming an unlocked “sugar” residue. Preferably thegapmer comprises a (poly)nucleotide sequence of formula (5′ to 3′),W-X-Y, or optionally W-X-Y-Z or V-W-X-Y, wherein: region W (W) (5′region) consists or comprises at least one nucleotide analogue, such asat least one LNA unit, such as from 1-6 nucleotide analogues, such asLNA units, and; region X (X) consists or comprises at least fiveconsecutive nucleotides which are capable of recruiting RNAse (whenformed in a duplex with a complementary RNA molecule, such as the mRNAtarget), such as DNA nucleotides, and; region Y (Y) (3′ region) consistsor comprises at least one nucleotide analogue, such as at least one LNAunit, such as from 1-6 nucleotide analogues, such as LNA units, and;region V (V) and/or region Z (Z), when present consists or comprises 1,2 or 3 nucleotide units, such as DNA nucleotides.

In some embodiments, region W consists of 1, 2, 3, 4, 5 or 6 nucleotideanalogues, such as LNA units, such as from 2-5 nucleotide analogues,such as 2-5 LNA units, such as 3 or 4 nucleotide analogues, such as 2, 3or 4 LNA units.

In some embodiments, region Y consists of 1, 2, 3, 4, 5 or 6 nucleotideanalogues, such as LNA units, such as from 2-5 nucleotide analogues,such as 2-5 LNA units, such as 2, 3 or 4 nucleotide analogues, such as 3or 4 LNA units.

In some embodiments, region X consists or comprises 5, 6, 7, 8, 9, 10,11 or 12 consecutive nucleotides which are capable of recruiting RNAse,or from 6-10, or from 7-9, such as 8 consecutive nucleotides which arecapable of recruiting RNAse.

In some embodiments region X consists or comprises at least one DNAnucleotide unit, such as 1-12 DNA units, preferably from 4-12 DNA units,more preferably from 6-10 DNA units, such as from 7-10 DNA units, mostpreferably 8, 9 or 10 DNA units.

In some embodiments, region W consist of 2, 3 or 4 nucleotide analogues,such as LNA, region X consists of 7, 8, 9 or 10 DNA units, and region Yconsists of 2, 3 or 4 nucleotide analogues, such as LNA. Such designsinclude (W-X-Y) 3-10-3, 3-10-4, 4-10-3, 3-9-3, 3-9-4, 4-9-3, 3-8-3,3-8-4, 4-8-3, 3-7-3, 3-7-4, 4-7-3, 3-10-2, 3-9-2, 3-8-2, 3-7-2, 4-10-2,4-9-2, 4-8-2, 4-7-2; and may further include region V, which may have 1,2 or 3 nucleotide units, such as DNA units and/or region Z, which mayhave 1, 2 or 3 nucleotide units, such as DNA units.

Further gapmer designs are disclosed in WO2004/046160, which is herebyincorporated by reference. WO2008/113832, which claims priority fromU.S. provisional application 60/977,409 hereby incorporated byreference, refers to ‘shortmer’ gapmer oligomers. In some embodiments,oligomers presented here may be such shortmer gapmers.

In some embodiments the oligomer is consisting of a contiguousnucleotide sequence of a total of 10, 11, 12, 13 or 14 nucleotide units,wherein the contiguous nucleotide sequence is of formula (5′-3′), W-X-Y,or optionally W-X-Y-Z or V-W-X-Y, wherein; W consists of 1, 2 or 3nucleotide analogue units, such as LNA units; X consists of 7, 8 or 9contiguous nucleotide units which are capable of recruiting RNAse whenformed in a duplex with a complementary RNA molecule (such as a mRNAtarget); and Y consists of 1, 2 or 3 nucleotide analogue units, such asLNA units. When present, V consists of 1 or 2 DNA units. When present, Zconsists of 1 or 2 DNA units.

In some embodiments W consists of 1 LNA unit. In some embodiments Wconsists of 2 LNA units. In some embodiments W consists of 3 LNA units.

In some embodiments Y consists of 1 LNA unit. In some embodiments Yconsists of 2 LNA units. In some embodiments Y consists of 3 LNA units.

In some embodiments X consists of 7 nucleotide units. In someembodiments X consists of 8 nucleotide units. In some embodiments Xconsists of 9 nucleotide units. In certain embodiments, region Xconsists of 10 nucleoside units. In certain embodiments, region Xcomprises 1-10 DNA units. In some embodiments X comprises from 1-9 DNAunits, such as 2, 3, 4, 5, 6, 7, 8 or 9 DNA units. In some embodiments Xconsists of DNA units. In some embodiments X comprises at least one LNAunit which is in the alpha-L configuration, such as 2, 3, 4, 5, 6, 7, 8or 9 LNA units in the alpha-L-configuration. In some embodiments Xcomprises at least one alpha-L-oxy LNA unit or wherein all the LNA unitsin the alpha-L-configuration are alpha-L-oxy LNA units.

In some embodiments the number of nucleotides present in W-X-Y areselected from the group consisting of (nucleotide analogue units—regionX—nucleotide analogue units): 1-8-1, 1-8-2, 2-8-1, 2-8-2, 3-8-3, 2-8-3,3-8-2, 4-8-1, 4-8-2, 1-8-4, 2-8-4, or; 1-9-1, 1-9-2, 2-9-1, 2-9-2,2-9-3, 3-9-2, 1-9-3, 3-9-1, 4-9-1, 1-9-4, or; 1-10-1, 1-10-2, 2-10-1,2-10-2, 1-10-3, 3-10-1, 3-10-2, 2-10-3, 3-10-3.

In some embodiments the number of nucleotides in W-X-Y are selected fromthe group consisting of: 2-7-1, 1-7-2, 2-7-2, 3-7-3, 2-7-3, 3-7-2,3-7-4, and 4-7-3; in some embodiments preferred motifs are 3-10-3,3-9-3, 3-8-3, 3-8-2.

In certain embodiments, each of regions W and Y consists of 2 or 3 LNAunits, and region X consists of 8 or 9 or 10 nucleoside units,preferably DNA units.

In some embodiments both W and Y consist of 2 or 3 LNA units each, and Xconsists of 8 or 9 nucleotide units, preferably DNA units.

In various embodiments, other gapmer designs include those where regionsW and/or Y consists of 3, 4, 5 or 6 nucleoside analogues, such as unitscontaining a 2′-O-methoxyethyl-ribose sugar (2′-MOE) or units containinga 2′-fluoro-deoxyribose sugar, and region X consists of 8, 9, 10, 11 or12 nucleosides, such as DNA units, where regions W-X-Y have 3-9-3,3-10-3, 5-10-5 or 4-12-4 units.

Further gapmer designs are disclosed in WO 2007/146511A2, herebyincorporated by reference.

Accordingly, the oligomer or the oligomer component of the oligomerconjugate may comprise at least 1, at least 2 or at least 3 modifiednucleotides at the 5′ end of the above sequences. For example, one ormore preferably all of the modified nucleotides may be LNA units.

The oligomer or the oligomer component of the oligomer conjugate maycomprise at least 1, at least 2 or at least 3 modified nucleotides atthe 3′ end of the above sequences. For example, one or more preferablyall of the modified nucleotides may be LNA units.

The oligomer or the oligomer component of the oligomer conjugate maycomprise at least 1, at least 2 or at least 3 modified nucleotides atthe 5′ and/or 3′ end of the sequences disclosed herein. For example, oneor more preferably all of the modified nucleotides may be LNA units.

For certain embodiments, the oligomer or the oligomer component of theoligomer conjugate may comprise at least 2 or at least 3 modifiednucleotides at the 5′ and/or 3′ end of the sequences disclosed herein.For example, two or more preferably all of the nucleotides may be LNAunits.

For certain embodiments, the oligomer or the oligomer component of theoligomer conjugate may comprise at least 3 modified nucleotides at the5′ and/or 3′ end of the sequences disclosed herein. For example, threeor more preferably all of the modified nucleotides may be LNA units.

For certain embodiments, the oligomer or the oligomer component of theoligomer conjugate may comprise 3 modified nucleotides at the 5′ and/or3′ end of the sequences disclosed herein. For example the modifiednucleotides may be LNA units.

For certain embodiments, the oligomer or the oligomer component of theoligomer conjugate may comprise 3 modified nucleotides at the 5′ and atthe 3′ end of the sequences disclosed herein. For example one or morepreferably all of the modified nucleotides may be LNA units.

For some embodiments, the oligomer may comprise an additional CAdinucleotide motif.

In certain aspects, preferably the oligomer is not GAGGCATAGCAGCAGG (SEQID NO: 102).

For certain embodiments of the invention, the oligomer or oligomercomponent of the oligomer conjugate comprises any one of the motifs:2-8-2, 3-8-3, 2-8-3, 3-8-2, 2-9-2, 3-9-3, 2-9-3, 3-9-2, 2-10-2, 3-10-3,3-10-2, 2-10-3 wherein the first number is the number of LNA units in anLNA wing region, the second number is the number of nucleotides in thegap region, and the third number is the number of LNA units in an LNAwing region.

For certain embodiments of the invention, the oligomer or oligomercomponent of the oligomer conjugate comprises any one of the motifs3-8-3, 3-8-2, 3-9-3, 3-9-2, 3-10-3, 3-10-2 wherein the first number isthe number of LNA units in an LNA wing region, the second number is thenumber of nucleotides in the gap region, and the third number is thenumber of LNA units in an LNA wing region.

For certain embodiments, the invention provides an oligomer or anoligomer conjugate as herein defined wherein the oligomer or oligomercomponent of the oligomer conjugate is a gapmer, and wherein the overallsequence comprises at least 6, preferably at least 7, preferably atleast 8, preferably at least 9, preferably at least 10 units, preferablyat least 11 units, preferably at least 12 units that are at least 80%,at least 85%, at least 90%, at least 95%, at least 98% identical to aregion corresponding to part of the HBV HBx gene, such as part of SEQ IDNo. 1, or part of the HBsAg gene, such as SEQ ID No. 2, or to thereverse complement of a target region of a nucleic acid which encodes aHBV HBx or HBV HBsAg.

For certain embodiments, the invention provides an oligomer or anoligomer conjugate as herein defined wherein the oligomer or oligomercomponent of the oligomer conjugate is a gapmer, and wherein thesequence of the central region comprises at least 6, preferably at least7, preferably at least 8, preferably at least 9, preferably at least 10units, that are at least 80%, at least 85%, at least 90%, at least 95%,at least 98% identical to a region corresponding to part of the HBV HBxgene, such as part of SEQ ID No. 1, or part of the HBsAg gene, such asSEQ ID No. 2, or to the reverse complement of a target region of anucleic acid which encodes a HBV HBx or HBV HBsAg.

For certain embodiments of the invention, the oligomer or the oligomercomponent of the oligomer conjugate may be based on a sequence selectedfrom the group consisting of any one or more of:

(SEQ ID NO: 13) GCGTAAAGAGAGG; (SEQ ID NO: 11) GCGTAAAGAGAGGT;(SEQ ID NO: 20) AGCGAAGTGCACACG; (SEQ ID NO: 26) AGGTGAAGCGAAGTG;(SEQ ID NO 18) AGCGAAGTGCACACGG; (SEQ ID NO: 7) CGAACCACTGAACA;(SEQ ID NO 4) GAACCACTGAACAAA; (SEQ ID NO 5) CGAACCACTGAACAAA;(SEQ ID NO 6) CGAACCACTGAACAA; (SEQ ID NO 8) CGAACCACTGAAC (SEQ ID NO 9)CCGCAGTATGGATCG (SEQ ID NO: 10) CGCAGTATGGATC; (SEQ ID NO 12)CGCGTAAAGAGAGGT; (SEQ ID NO 14) AGAAGGCACAGACGG; (SEQ ID NO 15)GAGAAGGCACAGACGG (SEQ ID NO 16) GAAGTGCACACGG; (SEQ ID NO 17)GCGAAGTGCACACGG; (SEQ ID NO 19) CGAAGTGCACACG; (SEQ ID NO 27)AGGTGAAGCGAAGT; and (SEQ ID NO: 852) TAGTAAACTGAGCCA,wherein 1, 2, 3 or 4 of the three to four 5′ terminal nucleotides aremodified nucleotides, for example LNA units; and 1, 2, 3 or 4 of thethree to four 3′ terminal nucleotides are modified nucleotides, forexample LNA units.

For certain embodiments of the invention, the oligomer or the oligomercomponent of the oligomer conjugate may be based on a sequence selectedfrom the group consisting of any one or more of:

(SEQ ID NO: 13) GCGTAAAGAGAGG; (SEQ ID NO: 20) AGCGAAGTGCACACG;(SEQ ID NO: 11) GCGTAAAGAGAGGT; (SEQ ID NO: 26) AGGTGAAGCGAAGTG;(SEQ ID NO 18) AGCGAAGTGCACACGG; (SEQ ID NO: 7) CGAACCACTGAACA;(SEQ ID NO 4) GAACCACTGAACAAA; (SEQ ID NO 5) CGAACCACTGAACAAA;(SEQ ID NO 6) CGAACCACTGAACAA; (SEQ ID NO 8) CGAACCACTGAAC (SEQ ID NO 9)CCGCAGTATGGATCG (SEQ ID NO: 10) CGCAGTATGGATC; (SEQ ID NO 12)CGCGTAAAGAGAGGT; (SEQ ID NO 14) AGAAGGCACAGACGG; (SEQ ID NO 15)GAGAAGGCACAGACGG (SEQ ID NO 16) GAAGTGCACACGG; (SEQ ID NO 17)GCGAAGTGCACACGG; (SEQ ID NO 19) CGAAGTGCACACG; (SEQ ID NO 27)AGGTGAAGCGAAGT; and (SEQ ID NO: 852) TAGTAAACTGAGCCA,

wherein

-   -   2 or 3 of the three 5′ terminal nucleotides are modified        nucleotides, for example LNA units;    -   2 or 3 of the three 3′ terminal nucleotides are modified        nucleotides, for example LNA units.

For certain embodiments of the invention, the oligomer or the oligomercomponent of the oligomer conjugate may be based on a sequence selectedfrom the group consisting of any one or more of:

(SEQ ID NO: 13) GCGTAAAGAGAGG; (SEQ ID NO: 11) GCGTAAAGAGAGGT;(SEQ ID NO: 20) AGCGAAGTGCACACG; (SEQ ID NO: 26) AGGTGAAGCGAAGTG;(SEQ ID NO 18) AGCGAAGTGCACACGG; (SEQ ID NO: 7) CGAACCACTGAACA;(SEQ ID NO 4) GAACCACTGAACAAA; (SEQ ID NO 5) CGAACCACTGAACAAA;(SEQ ID NO 6) CGAACCACTGAACAA; (SEQ ID NO 8) CGAACCACTGAAC (SEQ ID NO 9)CCGCAGTATGGATCG (SEQ ID NO: 10) CGCAGTATGGATC; (SEQ ID NO 12)CGCGTAAAGAGAGGT; (SEQ ID NO 14) AGAAGGCACAGACGG; (SEQ ID NO 15)GAGAAGGCACAGACGG (SEQ ID NO 16) GAAGTGCACACGG; (SEQ ID NO 17)GCGAAGTGCACACGG; (SEQ ID NO 19) CGAAGTGCACACG; (SEQ ID NO 27)AGGTGAAGCGAAGT; and (SEQ ID NO: 852) TAGTAAACTGAGCCA,wherein

-   -   the three 5′ terminal nucleotides are modified nucleotides, for        example LNA units;    -   2 or 3 of the three 3′ terminal nucleotides are modified        nucleotides, for example LNA units.

For certain embodiments of the invention, the oligomer or the oligomercomponent of the oligomer conjugate may be based on a sequence selectedfrom the group consisting of any one or more of:

(SEQ ID NO: 303) GCGtaaagagaGG; (SEQ ID NO: 301) GCGtaaagagaGGT;(SEQ ID NO: 618) GCGtaaagagAGG; (SEQ ID NO: 310) AGCgaagtgcacACG(SEQ ID NO: 668) AGgtgaagcgaAGTG; (SEQ ID NO: 308) AGCgaagtgcacaCGG;(SEQ ID NO: 297) CGAaccactgaACA; (SEQ ID NO: 300) CGCagtatggaTC;(SEQ ID NO: 315) AGGtgaagcgaagTGC (SEQ ID NO: 316) AGGtgaagcgaaGTG;(SEQ ID NO: 294) GAAccactgaacAAA; (SEQ ID NO: 295) CGAaccactgaacAAA;(SEQ ID NO: 296) CGAaccactgaaCAA; (SEQ ID NO: 298) CGAaccactgaAC;(SEQ ID NO: 299) CCGcagtatggaTCG; (SEQ ID NO: 302) CGCgtaaagagaGGT;(SEQ ID NO: 304) AGAaggcacagaCGG; (SEQ ID NO: 305) GAGaaggcacagaCGG;(SEQ ID NO: 306) GAAgtgcacacGG; (SEQ ID NO: 307) GCGaagtgcacaCGG;(SEQ ID NO: 309) CGAagtgcacaCG; (SEQ ID NO: 585) GAAccactgaaCAAA;(SEQ ID NO: 588) CGAAccactgaacAAA (SEQ ID NO: 628) GAAgtgcacaCGG;(SEQ ID NO: 678) TAGtaaactgagCCA; (SEQ ID NO: 600) CGAaccactgAAC;(SEQ ID NO: 317) AGGtgaagcgaAGT; and (SEQ ID NO: 597) CGAaccactgAACA,wherein upper case letters denote nucleotides which may be modifiednucleotides, for example LNA units.

For certain embodiments of the invention, the oligomer or the oligomercomponent of the oligomer conjugate may be based on a sequence selectedfrom the group consisting of any one or more of:

(SEQ ID NO: 303) 5′-AM-C6 ca

t_(s)a_(s)a_(s)a_(s)g_(s)a_(s)g_(s)a_(s)

-3′ (SEQ ID NO: 301) 5′-AM-C6 ca

t_(s)a_(s)a_(s)a_(s)g_(s)a_(s)g_(s)a_(s)

-3′ (SEQ ID NO: 618) 5′-AM-C6 ca

_(s)t_(s)a_(s)a_(s)a_(s)g_(s)a_(s)g_(s)

-3′ (SEQ ID NO: 310) 5′-AM-C6 ca

g_(s)a_(s)a_(s)g_(s)t_(s)g_(s)c_(s)a_(s)c_(s)

-3′ (SEQ ID NO: 668) 5′-AM-C6 ca

_(s)g_(s)t_(s)g_(s)a_(s)a_(s)g_(s)c_(s)g_(s)a_(s)

-3′ (SEQ ID NO: 308) 5′-AM-C6 ca

g_(s)a_(s)a_(s)g_(s)t_(s)g_(s)c_(s)a_(s)c_(s)a_(s)

-3′ (SEQ ID NO: 294) 5′-AM-C6 ca

c_(s)c_(s)a_(s)c_(s)t_(s)g_(s)a_(s)a_(s)c_(s)

-3′ (SEQ ID NO: 295) 5′-AM-C6 ca

a_(s)c_(s)c_(s)a_(s)c_(s)t_(s)g_(s)a_(s)a_(s)c_(s)

-3′ (SEQ ID NO: 296) 5′-AM-C6 ca

a_(s)c_(s)c_(s)a_(s)c_(s)t_(s)g_(s)a_(s)a_(s)

-3′ (SEQ ID NO: 299) 5′-AM-C6 ca

c_(s)a_(s)g_(s)t_(s)a_(s)t_(s)g_(s)g_(s)a_(s)

-3′ (SEQ ID NO: 302) 5′-AM-C6 ca

g_(s)t_(s)a_(s)a_(s)a_(s)g_(s)a_(s)g_(s)a_(s)

-3′ (SEQ ID NO: 304) 5′-AM-C6 ca

a_(s)g_(s)g_(s)c_(s)a_(s)c_(s)a_(s)g_(s)a_(s)

-3′ (SEQ ID NO: 305) 5′-AM-C6 ca

a_(s)a_(s)g_(s)g_(s)c_(s)a_(s)c_(s)a_(s)g_(s)a_(s)

-3′ (SEQ ID NO: 307) 5′-AM-C6 ca

a_(s)a_(s)g_(s)t_(s)g_(s)c_(s)a_(s)c_(s)a_(s)

-3′ (SEQ ID NO: 315) 5′-AM-C6 ca

t_(s)g_(s)a_(s)a_(s)g_(s) ^(m)c_(s)g_(s)a_(s)a_(s)g_(s)

-3′ (SEQ ID NO: 316) 5′-AM-C6 ca

t_(s)g_(s)a_(s)a_(s)g_(s) ^(m)c_(s)g_(s)a_(s)a_(s)

-3′ (SEQ ID NO: 297) 5′-AM-C6 ca

a_(s)c_(s)c_(s)a_(s)c_(s)t_(s)g_(s)a_(s)

-3′ (SEQ ID NO: 298) 5′-AM-C6 ca

a_(s)c_(s)c_(s)a_(s)c_(s)t_(s)g_(s)a_(s)

-3′ (SEQ ID NO: 300) 5′-AM-C6 ca

a_(s)g_(s)t_(s)a_(s)t_(s)g_(s)g_(s)a_(s)

-3′ (SEQ ID NO: 306) 5′-AM-C6 ca

g_(s)t_(s)g_(s)c_(s)a_(s)c_(s)a_(s) ^(m)c_(s)

-3′ (SEQ ID NO: 309) 5′-AM-C6 ca

a_(s)g_(s)t_(s)g_(s)c_(s)a_(s)c_(s)a_(s)

-3′ (SEQ ID NO: 317) 5′-AM-C6 ca

t_(s)g_(s)a_(s)a_(s)g_(s) ^(m)c_(s)g_(s)a_(s)

-3′ (SEQ ID NO: 585) 5′-AM-C6 ca

_(s)c_(s)c_(s)a_(s)c_(s)t_(s)g_(s)a_(s)a_(s)

-3′ (SEQ ID NO: 588) 5′-AM-C6 ca^(m)

_(s)c_(s)c_(s)a_(s)c_(s)t_(s)g_(s)a_(s)a_(s)c_(s)

-3′ (SEQ ID NO: 628) 5′-AM-C6 ca

_(s)g_(s)t_(s)g_(s)c_(s)a_(s)c_(s)a_(s)

-3′ (SEQ ID NO: 678) 5′-AM-C6 ca

_(s)t_(s)a_(s)a_(s)a_(s)c_(s)t_(s)g_(s)a_(s)g_(s)

_(s)3′ (SEQ ID NO: 600) 5′-AM-C6 ca

_(s)a_(s)c_(s)c_(s)a_(s)c_(s)t_(s)g_(s)

-3′ (SEQ ID NO: 597) 5′-AM-C6 ca

_(s)a_(s)c_(s)c_(s)a_(s)c_(s)t_(s)g_(s)

-3′wherein

-   -   uppercase letters denote beta-D-oxy-LNA units;    -   lowercase letters denote DNA units;    -   the subscript “s” denotes a phosphorothioate linkage;    -   superscript m denotes a DNA or beta-D-oxy-LNA unit containing a        5-methylcytosine base;    -   AM-C6 is an amino-C6 linker; wherein the 5′ terminal group        “AM-C6 c a” is optional.

AM-C6 is an amino-C6 linker: 6-aminohexanol in the 5′-end of theoligonucleotide linked via a phosphodiester or phorphothioate

Accordingly, for certain embodiments of the invention, the oligomer orthe oligomer component of the oligomer conjugate may be based on asequence selected from the group consisting of any one or more of:

(SEQ ID NO: 303) G^(m)CGtaaagagaGG; (SEQ ID NO: 301) G^(m)CGtaaagagaGGT;(SEQ ID NO: 618) G^(m)CGtaaagagAGG; (SEQ ID NO: 310)AG^(m)CgaagtgcacA^(m)CG (SEQ ID NO: 668) AGgtgaag^(m)cgaAGTG;(SEQ ID NO: 308) AG^(m)Cgaagtgcaca^(m)CGG; (SEQ ID NO: 297)^(m)CGAaccactgaA^(m)CA; (SEQ ID NO: 300) ^(m)CG^(m)CagtatggaT^(m)C;(SEQ ID NO: 315) AGGtgaagcgaagTGC; (SEQ ID NO: 316) AGGtgaag^(m)cgaaGTG;(SEQ ID NO: 294) GAAccactgaacAAA; (SEQ ID NO: 295) ^(m)CGAaccactgaacAAA;(SEQ ID NO: 296) ^(m)CGAaccactgaa^(m)CAA; (SEQ ID NO: 298)^(m)CGAaccactgaA^(m)C; (SEQ ID NO: 299) ^(m)C^(m)CGcagtatggaT^(m)CG;(SEQ ID NO: 302) ^(m)CG^(m)CgtaaagagaGGT; (SEQ ID NO: 304)AGAaggcacaga^(m)CGG; (SEQ ID NO: 305) GAGaaggcacaga^(m)CGG;(SEQ ID NO: 306) GAAgtgcaca^(m)cGG; (SEQ ID NO: 307)G^(m)CGaagtgcaca^(m)CGG; (SEQ ID NO: 309) ^(m)CGAagtgcaca^(m)CG;(SEQ ID NO: 585) GAAccactgaa^(m)CAAA; (SEQ ID NO: 588)^(m)CGAAccactgaacAAA (SEQ ID NO: 628) GAAgtgcaca^(m)CGG;(SEQ ID NO: 678) TAGtaaactgag^(m)C^(m)CA; (SEQ ID NO: 600)^(m)CGAaccactgAA^(m)C; (SEQ ID NO: 317) AGGtgaag^(m)cgaAGT; and(SEQ ID NO: 597) ^(m)CGAaccactgAA^(m)CA,wherein

-   -   uppercase letters denote beta-D-oxy-LNA units;    -   lowercase letters denote DNA units;    -   all internucleoside linkages are phosphorothioate linkages;    -   superscript m denotes a DNA or beta-D-oxy-LNA unit containing a        5-methylcytosine base.

In certain preferred aspects, the oligomer or oligomer component of theoligomer conjugate comprises any one of the motifs: 3-10-3, 3-10-2,3-9-3, 3-9-2, 3-8-3, 3-8-2 wherein the first number is the number ofmodified nucleotides in the wing region, preferably at least one beingan LNA unit, preferably all being an LNA unit, the second number is thenumber of nucleotides in the gap region, and the third number is thenumber of modified nucleotides in the wing region, preferably at leastone being an LNA unit, preferably all being an LNA unit.

As indicated, the oligomer of the invention may comprise or may be agapmer. Alternatively expressed, a gapmer oligomer is an oligomer whichcomprises a contiguous stretch of nucleotides which is capable ofrecruiting an RNAse, such as RNAseH, such as a region of at least 6 or 7DNA nucleotides, referred to herein in as region GH (GH), wherein regionGH is flanked both 5′ and 3′ by regions of affinity enhancing nucleotideanalogues, such as from 1-6 nucleotide analogues 5′ and 3′ to thecontiguous stretch of nucleotides which is capable of recruitingRNAse—these regions are referred to as regions GX′ (GX′) and GZ′ (GZ′).Regions GX, GH and GZ correspond to regions W, X and Y, respectively.

In some embodiments, the units which are capable of recruiting RNAse areselected from the group consisting of DNA units, alpha-L-LNA units, C4′alkylayted DNA units (see PCT/EP2009/050349 and Vester et al., Bioorg.Med. Chem. Lett. 18 (2008) 2296-2300, hereby incorporated by reference),and UNA (unlinked nucleic acid) nucleotides (see Fluiter et al., Mol.Biosyst., 2009, 10, 1039 hereby incorporated by reference). UNA isunlocked nucleic acid, typically where the C2-C3 C—C bond of the ribosehas been removed, forming an unlocked “sugar” residue. Preferably thegapmer comprises a (poly)nucleotide sequence of formula (5′ to 3′),GX′-GH-GZ′, wherein; region GX′ (GX′) (5′ region) consists or comprisesof at least one nucleotide analogue, such as at least one BNA (e.g. LNA)unit, such as from 1-6 nucleotide analogues, such as BNA (e.g. LNA)units, and; region GH (H) consists or comprises of at least fiveconsecutive nucleotides which are capable of recruiting RNAse (whenformed in a duplex with a complementary RNA molecule, such as the mRNAtarget), such as DNA nucleotides, and; region GZ′ (GZ′) (3′region)consists or comprises of at least one nucleotide analogue, such as atleast one BNA (e.g LNA unit), such as from 1-6 nucleotide analogues,such as BNA (e.g. LNA) units.

In some embodiments, region GX′ consists of 1, 2, 3, 4, 5 or 6nucleotide analogues, such as BNA (e.g. LNA) units, such as from 2-5nucleotide analogues, such as 2-5 LNA units, such as 3 or 4 nucleotideanalogues, such as 3 or 4 LNA units; and/or region GZ′ consists of 1, 2,3, 4, 5 or 6 nucleotide analogues, such as BNA (e.g. LNA) units, such asfrom 2-5 nucleotide analogues, such as 2-5 BNA (e.g. LNA units), such as3 or 4 nucleotide analogues, such as 3 or 4 BNA (e.g. LNA) units.

In some embodiments GH consists or comprises of 5, 6, 7, 8, 9, 10, 11 or12 consecutive nucleotides which are capable of recruiting RNAse, orfrom 6-10, or from 7-9, such as 8 consecutive nucleotides which arecapable of recruiting RNAse. In some embodiments region GH consists orcomprises at least one DNA nucleotide unit, such as 1-12 DNA units,preferably from 4-12 DNA units, more preferably from 6-10 DNA units,such as from 7-10 DNA units, most preferably 8, 9 or 10 DNA units.

In some embodiments region GX′ consist of 3 or 4 nucleotide analogues,such as BNA (e.g. LNA), region X′ consists of 7, 8, 9 or 10 DNA units,and region Z′ consists of 3 or 4 nucleotide analogues, such as BNA (e.g.LNA). Such designs include (GX′-GH-GZ′) 3-10-3, 3-10-4, 4-10-3, 3-9-3,3-9-4, 4-9-3, 3-8-3, 3-8-4, 4-8-3, 3-7-3, 3-7-4, 4-7-3.

In some embodiments the oligomer, e.g. region GX′, is consisting of acontiguous nucleotide sequence of a total of 10, 11, 12, 13 or 14nucleotide units, wherein the contiguous nucleotide sequence comprisesor is of formula (5′-3′), GX′-GH-GZ′ wherein; GX′ consists of 1, 2 or 3nucleotide analogue units, such as BNA (e.g. LNA) units; GH consists of7, 8 or 9 contiguous nucleotide units which are capable of recruitingRNAse when formed in a duplex with a complementary RNA molecule (such asa mRNA target); and GZ′ consists of 1, 2 or 3 nucleotide analogue units,such as BNA (e.g. LNA) units.

In some embodiments GX′ consists of 1 BNA (e.g. LNA) unit. In someembodiments GX′ consists of 2 BNA (e.g. LNA) units. In some embodimentsGX′ consists of 3 BNA (e.g. LNA) units. In some embodiments GZ′ consistsof 1 BNA (e.g. LNA) units. In some embodiments GZ′ consists of 2 BNA(e.g. LNA) units. In some embodiments GZ′ consists of 3 BNA (e.g. LNA)units. In some embodiments GH consists of 7 nucleotide units. In someembodiments GH consists of 8 nucleotide units. In some embodiments GHconsists of 9 nucleotide units. In certain embodiments, region GHconsists of 10 nucleoside units. In certain embodiments, region GHconsists or comprises 1-10 DNA units. In some embodiments GH comprisesof from 1-9 DNA units, such as 2, 3, 4, 5, 6, 7, 8 or 9 DNA units. Insome embodiments GH consists of DNA units. In some embodiments GHcomprises of at least one BNA unit which is in the alpha-Lconfiguration, such as 2, 3, 4, 5, 6, 7, 8 or 9 LNA units in thealpha-L-configuration. In some embodiments GH comprises of at least onealpha-L-oxy BNA/LNA unit or wherein all the LNA units in thealpha-L-configuration are alpha-L-oxy LNA units. In some embodiments thenumber of nucleotides present in GX′-GH-GZ′ are selected from the groupconsisting of (nucleotide analogue units—region GH—nucleotide analogueunits): 1-8-1, 1-8-2, 2-8-1, 2-8-2, 3-8-3, 2-8-3, 3-8-2, 4-8-1, 4-8-2,1-8-4, 2-8-4, or; 1-9-1, 1-9-2, 2-9-1, 2-9-2, 2-9-3, 3-9-2, 1-9-3,3-9-1, 4-9-1, 1-9-4, or; 1-10-1, 1-10-2, 2-10-1, 2-10-2, 1-10-3, 3-10-1.In some embodiments the number of nucleotides in GX′-GH-GZ′ are selectedfrom the group consisting of: 2-7-1, 1-7-2, 2-7-2, 3-7-3, 2-7-3, 3-7-2,3-7-4, and 4-7-3. In certain embodiments, each of regions GX′ and GHconsists of three BNA (e.g. LNA) units, and region GH consists of 8 or 9or 10 nucleoside units, preferably DNA units. In some embodiments bothGX′ and GZ′ consists of two BNA (e.g. LNA) units each, and GH consistsof 8 or 9 nucleotide units, preferably DNA units. In variousembodiments, other gapmer designs include those where regions GX′ and/orGZ′ consists of 3, 4, 5 or 6 nucleoside analogues, such as unitscontaining a 2′-O-methoxyethyl-ribose sugar (2′-MOE) or units containinga 2′-fluoro-deoxyribose sugar, and region H consists of 8, 9, 10, 11 or12 nucleosides, such as DNA units, where regions GX′-GH-GZ′ have 3-9-3,3-10-3, 5-10-5 or 4-12-4 units.

BNA and LNA Gapmers: The terms BNA and LNA are used interchangeably. ABNA gapmer is a gapmer oligomer (region GA) which comprises at least oneBNA nucleotide. A LNA gapmer is a gapmer oligomer (region GA) whichcomprises at least one LNA nucleotide.

Internucleotide Linkages

The units of the oligomers and oligomer conjugates described herein arecoupled together via linkage groups. Suitably, each unit is linked tothe 3′ adjacent unit via a linkage group.

The person having ordinary skill in the art would understand that, inthe context of the present invention, the 5′ unit at the end of anoligomer does not comprise a 5′ linkage group, although it may or maynot comprise a 5′ terminal group.

The nucleotides of the oligomer of the invention or contiguousnucleotides sequence thereof are coupled together via linkage groups.Suitably each nucleotide is linked to the 3′ adjacent nucleotide via alinkage group.

Optionally, the oligonucleotide of the invention or the oligonucleotideconjugate of the invention may comprise one or more linker groups and/orone or more brancher regions. In various embodiments, the linker groupsare internucleoside or internucleotide linkages.

Suitable internucleotide linkages include those listed withinWO2007/031091, for example the internucleotide linkages listed on thefirst paragraph of page 34 of WO2007/031091 (hereby incorporated byreference).

For some embodiments, it is preferred to modify the internucleotidelinkage from its normal phosphodiester to one that is more resistant tonuclease attack, such as phosphorothioate or boranophosphate—these two,being cleavable by RNase H, also allow that route of antisenseinhibition in reducing the expression of the target gene.

Suitable sulphur (S) containing internucleotide linkages as providedherein may be preferred. Phosphorothioate internucleotide linkages arealso preferred, particularly for the gap region (X) of gapmers.Phosphorothioate linkages may also be used for the flanking regions (Wand Y, and for linking W or Y to V or Z, and within region V or regionZ, as appropriate).

Regions W, X and Y, may however comprise internucleotide linkages otherthan phosphorothioate, such as phosphodiester linkages, particularly,for instance when the use of nucleotide analogues protects theinternucleotide linkages within regions W and Y from endo-nucleasedegradation—such as when regions W and Y comprise LNA nucleotides.

The internucleotide linkages in the oligomer may be phosphodiester,phosphorothioate or boranophosphate so as to allow RNase H cleavage oftargeted RNA. Phosphorothioate is preferred for improved nucleaseresistance and other reasons, such as ease of manufacture.

In one aspect of the oligomer of the invention, the nucleotides and/ornucleotide analogues are linked to each other by means ofphosphorothioate groups.

It is recognised that the inclusion of phosphodiester linkages, such asone or two linkages, into an otherwise phosphorothioate oligomer,particularly between or adjacent to nucleotide analogue units (typicallyin region W and or Y) can modify the bioavailability and/orbio-distribution of an oligomer—see WO2008/113832, hereby incorporatedby reference.

In some embodiments, such as the embodiments referred to above, wheresuitable and not specifically indicated, all remaining linkage groupsare either phosphodiester or phosphorothioate, or a mixture thereof.

In some embodiments all the internucleotide linkage groups arephosphorothioate.

When referring to specific gapmer oligonucleotide sequences, such as anyof those specific sequences provided herein it will be understood that,in various embodiments, when the linkages are phosphorothioate linkages,alternative linkages, such as those disclosed herein may be used, forexample phosphate (phosphodiester) linkages may be used, particularlyfor linkages between nucleotide analogues, such as LNA units. Likewise,when referring to specific gapmer oligonucleotide sequences, such as anyof those specific sequences provided herein, when the C residues areannotated as 5′methyl modified cytosine, in various embodiments, one ormore of the Cs present in the oligomer may be unmodified C residues.

WO09124238 refers to oligomeric compounds having at least one bicyclicnucleoside attached to the 3′ or 5′ termini by a neutral internucleosidelinkage. The oligomers of the invention may therefore have at least onebicyclic nucleoside attached to the 3′ or 5′ termini by a neutralinternucleoside linkage, such as one or more phosphotriester,methylphosphonate, MMI, amide-3, formacetal or thioformacetal. Theremaining linkages may be phosphorothioate.

Carrier

In some embodiments, the oligomer of the present invention is linked toone or more carrier components, which may be the same or different.

In some embodiments, the oligomer conjugate has or comprises thestructure:

-   -   Carrier component-L-First Oligomer Region        wherein L is an optional linker or brancher region or tether        molecule or bridging moiety. Preferably L is selected from a        physiologically labile linker (region PL) or an alternative        linker (Region E), or a combination of both.

The first oligomer region can be linked to the linker or carrier via the5′-end illustrated as follows:

-   -   Carrier component-L1-First Oligomer Region        -   wherein L1 is an optional linker or brancher region or            tether molecule or bridging moiety; or    -   Alternatively the first oligomer region can be linked to the        linker or carrier via the 3′-end illustrated as follows: First        Oligomer Region-L2-Carrier component        -   wherein L2 is an optional linker or brancher region or            tether molecule or bridging moiety.

For certain embodiments, preferably the oligomer conjugate has orcomprises the structure:

-   -   Carrier component-L1-First Oligomer Region    -   wherein L1 is an optional linker.

For certain embodiments, preferably Linker 1 is present.

For certain embodiments, preferably said carrier component is linked,preferably conjugated, to said first oligomer region.

For certain embodiments, preferably said carrier component is linked,preferably conjugated, to the 5′ end of said oligomer.

For certain embodiments, preferably said carrier component is linked,preferably conjugated, to the 5′ end of said oligomer by means of alinker group or a brancher region or tether molecule or bridging moiety.

For certain embodiments, preferably the linker group or the brancherregion is a physiologically labile linker group or a physiologicallylabile brancher region or physiologically labile tether molecule orphysiologically labile bridging moiety.

For certain embodiments, preferably the physiologically labile linkergroup is a nuclease susceptible linker, preferably a phosphodiesterlinker.

For certain embodiments the preferred L1 linker is composed of aphysiologically labile linker and a C2-C36 amino alkyl group, including,for example C6 to C12 amino alkyl groups. In a preferred embodiment theL1 linker is composed a PO linker and a C6 amino linker.

In some embodiments, the carrier component is selected from acarbohydrate conjugate or a lipophilic conjugate, or the carriercomponent comprises both a carbohydrate and a lipophilic conjugate.

The carbohydrate conjugate moiety may for example be selected from thegroup consisting of galactose, galactosamine, N-formyl-galactosamine,Nacetylgalactosamine, N-propionyl-galactosamine,N-n-butanoyl-galactosamine, and N-isobutanoylgalactose-amine. Preferablythe carbohydrate conjugate moiety is an asialoglycoprotein receptortargeting conjugate moiety. The lipophilic conjugate may be ahydrophobic group, such as a C16-20 hydrophobic group, a sterol,cholesterol. Other carbohydrate and lipophilic groups which may be usedare, for example, disclosed herein.

For some embodiments, the oligomer may comprise an additional CAdinucleotide motif. Preferably, in the context of the oligomerconjugate, the CA motif is positioned between the carrier component andthe oligomer. The CA motif preferably comprise a phosphidiester linkageand serves as a PO linker between the oligomer and the carriercomponent.

Hepatitis B Virus (HBV)

It is intended that all oligomers and oligomer conjugates as describedherein are capable of being used to treat a viral disorder, inparticular a disorder associated with HBV. Such uses form part of thepresent invention.

It is intended that all oligomers and oligomer conjugates as describedherein are capable of being used in the manufacture of a medicament totreat a viral disorder, in particular a disorder associated with HBV.Such uses form part of the present invention.

It is intended that all oligomers and oligomer conjugates as describedherein are capable of being administered to a subject as part of amethod for alleviating, curing or treating a viral disorder, inparticular a disorder associated with HBV. Such methods form part of thepresent invention.

It is intended that all oligomers and oligomer conjugates as describedherein are capable of comprising part of a pharmaceutical composition totreat a viral disorder, in particular a disorder associated with HBV.Such pharmaceutical compositions form part of the present invention.

Hepatitis B virus (HBV) is a species of the genus Orthohepadnavirus,which is a part of the Hepadnaviridae family. The virus causes thedisease hepatitis B. In addition to causing hepatitis, infection withHBV can lead to cirrhosis and hepatocellular carcinoma. It has also beensuggested that it may increase the risk of pancreatic cancer.

The virus is divided into four major serotypes (adr, adw, ayr, ayw)based on antigenic epitopes present on its envelope proteins, and intoeight genotypes (A-H) according to overall nucleotide sequence variationof the genome. The genotypes have a distinct geographical distributionand differences between genotypes affect the disease severity, courseand likelihood of complications, and response to treatment and possiblyvaccination. The genotypes differ by at least 8%. Type F which divergesfrom the other genomes by 14% is the most divergent type known. Type Ais prevalent in Europe, Africa and South-east Asia, including thePhilippines. Type B and C are predominant in Asia; type D is common inthe Mediterranean area, the Middle East and India; type E is localizedin sub-Saharan Africa; type F (or H) is restricted to Central and SouthAmerica. Type G has been found in France and Germany. Genotypes A, D andF are predominant in Brazil and all genotypes occur in the United Stateswith frequencies dependent on ethnicity.

HBV has a circular DNA genome, however, the DNA is not fullydouble-stranded as one end of the full length strand is linked to theviral DNA polymerase. The genome is 3020-3320 nucleotides long (for thefull length strand) and 1700-2800 nucleotides long (for the short lengthstrand).

There are four known genes encoded by the HBV genome (C, P, S, and X).The core protein is coded for by gene C (HBcAg), and its start codon ispreceded by an upstream in-frame AUG start codon from which the pre-coreprotein is produced. HBeAg is produced by proteolytic processing of thepre-core protein. The DNA polymerase is encoded by gene P.

HBx

The HBx polypeptide is a 154 residue protein which interferes withtranscription, signal transduction, cell cycle progress, proteindegradation, apoptosis and chromosomal stability in the host. It forms aheterodimeric complex with its cellular target protein (HBX interactingprotein: HBXIP), and this interaction dysregulates centrosome dynamicsand mitotic spindle formation. It interacts with DDB1 (Damaged DNABinding Protein 1) redirecting the ubiquitin ligase activity of theCUL4-DDB1 E3 complexes, which are intimately involved in theintracellular regulation of DNA replication and repair, transcriptionand signal transduction.

Although it lacks significant sequence identity with any knownvertebrate proteins, it is likely to have evolved from a DNAglycosylase. Transgenic mice expressing the X protein in liver are morelikely than the wild type to develop hepatocellular carcinoma. This isbecause the X protein promotes cell cycle progression while binding toand inhibiting tumour suppressor protein p53 from performing their role.Experimental observations also suggest that HBx protein increases TERTand telomerase activity, prolonging the lifespan of hepatocytes andcontributing to malignant transformation.

In a study purifying cancerous liver cells infected with HBV, the levelof expression of protein arginine methyltransferase 1 (PRMT1) was foundto be associated with changes in transcription due to themethyltransferase function of PRMT1. Overexpression causes a reductionin the number of HBV genes transcribed, while conversely, reducedexpression causes an increase. PRMT1 was also found to be recruited byHBV DNA during the replication process to regulate the transcriptionprocess. Increased HBx expression in turn leads to an inhibition ofPRMT1-mediated protein methylation, benefiting viral replication.

The HBx target sequence comprises sequences starting at the firstreported transcription start site at position 1196 to thepolyadenylation site at position 1941. The sequence from position 1196to 1941 of the U95551 sequence is presented as SEQ ID No. 1. The U95551sequence is presented as SEQ ID No. 3.

HBsAg

HBsAg (also known as Major surface antigen, HBV major surface antigen,HBV surface antigen and ‘5’) is the surface antigen of the HBV.

Gene S is the gene that codes for the surface antigen (HBsAg). The HBsAggene is one long open reading frame but contains three in frame “start”(ATG) codons that divide the gene into three sections, pre-S1, pre-S2,and S. Because of the multiple start codons, polypeptides of threedifferent sizes called large, middle, and small (pre-S1+pre-S2+S,pre-S2+S, or S) are produced.

The HBsAg is made up of three glycoproteins that are encoded by the samegene. The proteins are translated in the same reading frame but start ata different AUG start codon; thus, all have the same C-terminus. Thelargest protein is the L protein (42kd) and contained within this is theM glycoprotein. The S glycoprotein (27kD) is contained within the Mprotein. The HBsAg protein is also secreted into the patient's serumwhere it can be seen as spherical (mostly self-associated S protein) orfilamentous particles (also mostly S protein but with some L and M). Theformer are smaller than the true virus but the filaments can be quitelarge (several hundred nanometers).

S—HBsAg is 226 amino acid residues in length. It is an integral membraneglycoprotein which is anchored in the ER lipid bilayer through anamino-terminal transmembrane domain (TMD-I) between residues 4 and 24.It comprises a downstream cytosolic loop (CYL-I) between residues 24 and80, a second transmembrane domain (TMD-II) between residues 80 and 100,and an antigenic loop (AGL) encompassing residues 101 to 164, facing theER lumen (or the surface of extracellular particles). The carboxylterminus (residues 165 to 226) is predicted to contain two TMDs (TMD-IIIand -IV), located at positions 173 to 193 and 202 to 222, respectively,separated by a short sequence (residues 194 to 201) referred to here ascytosolic loop II (CYL-II) because it is predicted to reside at thecytosolic side of the ER membrane. The M-HBsAg protein sequence islonger than that of S—HBsAg by 55 residues (the pre-S2 domain) at itsamino terminus; it is coassembled with the latter in the viral envelopebut is dispensable for both morphogenesis and in vitro infectivity.L-HBsAg comprises the entire M polypeptide with an additionalamino-terminal extension (pre-S1) of 108 to 119 residues depending onthe HBV genotype. It has been described with two topologies, with theamino-terminal pre-S domain (pre-S1 plus pre-S2) being either cytosolicat the ER membrane (internal on secreted virions) or luminal (exposed atthe virion surface). The internal conformation is involved in recruitingthe nucleocapsid for virion assembly, whereas the external positioncorresponds to a receptor-binding function at viral entry.

All three envelope proteins are synthesized at the endoplasmic reticulum(ER) membrane, where they aggregate through protein-protein interactionsleading primarily to the secretion of empty S—HBsAg-coated subviralparticles (SVPs) It is only when L-HBsAg is present in the envelopeprotein aggregates at the ER membrane that the HBV nucleocapsid can berecruited in the budding complex and released as a mature virion. Owingto the overwhelming activity of S—HBsAg for self-assembly, in comparisonto that of L-HBsAg, HBV virion formation occurs only on rare occasions.

The HBsAg target sequence comprises sequences starting of thecircularized U95551 sequence from the transcription start site atposition 3158 to 3182 and from position 1 to the polyadenylation site atposition 1941. Thus, the HBsAg target sequence comprises sequences ofthe combined sequences from position 3158 to 3182 and position 1 to 1944of the circularized U95551 sequence. This target sequence is presentedas SEQ ID no. 2. The U95551 sequence is presented as SEQ ID No. 3.

Target

In a preferred aspect, suitably the oligomer or the oligomer conjugateof the invention is capable of modulating a target sequence in HBx orHBsAg of Hepatitis B Virus (HBV). In this regard, the oligomer of theinvention can affect the inhibition of a target sequence, typically in amammalian cell such as a human cell, such as a liver cell.

In some embodiments, the oligomer or the oligomer conjugate of theinvention binds to the target nucleic acid and modulates expression byat least 10% or 20% compared to the normal expression level, morepreferably at least a 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% comparedto the normal expression level (such as the expression level in theabsence of the oligomer(s) or oligomer conjugate(s)).

In one embodiment, the oligomer or the oligomer conjugate of theinvention is capable of down-regulating (e.g. inhibiting, reducing orremoving) expression of the HBx or HBsAg gene. In this regard, theoligomer of the invention can affect the inhibition of HBx or HBsAg.Such inhibition may typically occur in a mammalian cell such as a humancell, such as a liver cell. In some embodiments, the oligomers of theinvention bind to the target nucleic acid and affect inhibition ofexpression of at least 10% or 20% compared to the normal expressionlevel, more preferably at least a 30%, 40%, 50%, 60%, 70%, 80%, 90% or95% inhibition compared to the normal expression level (such as theexpression level in the absence of the oligomer(s) or oligomerconjugate(s)).

In some embodiments, such modulation is seen when using from 0.04 and 25nM, such as from 0.8 and 20 nM concentration of the compound of theinvention. In the same or a different embodiment, the modulation ofexpression is less than 100%, such as less than 98%, less than 95%, lessthan 90%, less than 80%, such as less than 70%. Modulation of expressionlevel may be determined by measuring protein levels, e.g. by the methodssuch as SDS-PAGE followed by western blotting using suitable antibodiesraised against the target protein. Alternatively, modulation ofexpression levels can be determined by measuring levels of mRNA, e.g. bynorthern blotting or quantitative RT-PCR. When measuring via mRNAlevels, the level of down-regulation when using an appropriate dosage,such as from 0.04 and 25 nM, such as from 0.8 and 20 nM concentration,is, in some embodiments, typically to a level of from 10-20% the normallevels in the absence of the compound, conjugate or composition of theinvention.

As illustrated herein the cells may be in vitro transfected cells. Theconcentration of the oligomer or the oligomer conjugate used may, insome embodiments, be 5 nM. The oligomer concentration used may, in someembodiments be 25 nM. The concentration of the oligomer or the oligomerconjugate used may, in some embodiments be 1 nM. It should be noted thatthe concentration of oligomer used to treat the cell is typicallyperformed in an in vitro cell assay, using transfection (Lipofecton), asillustrated in the examples. In the absence of a transfection agent, theconcentration of the oligomer or the oligomer conjugate required toobtain the down-regulation of the target is typically between 1 and 25μM, such as 5 μM.

The invention therefore provides a method of down-regulating orinhibiting the expression of HBx or HBsAg protein and/or mRNA in a cellwhich is expressing HBx or HBsAg protein and/or mRNA, said methodcomprising administering the oligomer or conjugate according to theinvention to said cell to down-regulate or inhibit the expression of HBxor HBsAg protein and/or mRNA in said cell. Suitably the cell is amammalian cell such as a human cell. The administration may occur, insome embodiments, in vitro. The administration may occur, in someembodiments, in vivo.

For certain embodiments, the oligomer or the oligomer component of theoligomer conjugate that targets HBx also binds to HBsAg.

Target Sequence

The oligomers or oligomer conjugates comprise or consist of a contiguousnucleotide sequence which corresponds to the reverse complement of anucleotide sequence present in the target sequence.

The target sequence may be a gene or a mRNA, such as a coding or anon-coding region of a gene or mRNA. For example, the target sequencemay be a coding or non-coding exon. The target sequence may comprise atleast part of an exon. The target sequence may be part of an exon, suchas within an exon.

In the practice of the present invention, the target sequence may besingle-stranded or double-stranded DNA or RNA; however, single-strandedDNA or RNA targets are preferred. It is understood that the targetsequence to which the antisense oligonucleotides of the invention aredirected include allelic forms of the targeted gene and thecorresponding mRNAs including splice variants. There is substantialguidance in the literature for selecting particular sequences forantisense oligonucleotides given a knowledge of the sequence of thetarget polynucleotide, e.g., Cook S. T. Antisense Drug Technology,Principles, Strategies, and Applications, Marcel Dekker, Inc, 2001;Peyman and Ulmann, Chemical Reviews, 90:543-584, 1990; and Crooke, Ann.Rev. Pharmacol. Toxicol., 32:329-376 (1992). mRNA targets may includethe 5′ cap site, tRNA primer binding site, the initiation codon site,the mRNA donor splice site, and the mRNA acceptor splice site.

Where the target polynucleotide sequence comprises a mRNA transcript,sequence complementary oligonucleotides can hybridize to any desiredportion of the transcript. Such oligonucleotides are, in principle,effective for inhibiting translation, and capable of inducing theeffects described herein. It is hypothesized that translation is mosteffectively inhibited by blocking the mRNA at a site at or near theinitiation codon. Thus, oligonucleotides may be complementary to the5′-region of mRNA transcript. Oligonucleotides may be complementary tothe mRNA, including the initiation codon (the first codon at the 5′ endof the translated portion of the transcript), or codons adjacent to theinitiation codon.

In one embodiment, the target sequence may have identity between HBVgenotypes A-H (described in detail below).

In one embodiment, the target sequence may have at least 80%, at least85%, at least 90%, at least 95%, at least 98% or at least 99% identitywith any one or more of the HBV genotypes A-H. For example, the targetsequence may have at least 80%, at least 85%, at least 90%, at least95%, at least 98% or at least 99% identity with HBV genotype A. Forexample, the target sequence may have at least 80%, at least 85%, atleast 90%, at least 95%, at least 98% or at least 99% identity with HBVgenotype B. For example, the target sequence may have at least 80%, atleast 85%, at least 90%, at least 95%, at least 98% or at least 99%identity with HBV genotype C. For example, the target sequence may haveat least 80%, at least 85%, at least 90%, at least 95%, at least 98% orat least 99% identity with HBV genotype D. For example, the targetsequence may have at least 80%, at least 85%, at least 90%, at least95%, at least 98% or at least 99% identity with HBV genotype E. Forexample, the target sequence may have at least 80%, at least 85%, atleast 90%, at least 95%, at least 98% or at least 99% identity with HBVgenotype F. For example, the target sequence may have at least 80%, atleast 85%, at least 90%, at least 95%, at least 98% or at least 99%identity with HBV genotype G. For example, the target sequence may haveat least 80%, at least 85%, at least 90%, at least 95%, at least 98% orat least 99% identity with HBV genotype H.

In one embodiment, the target sequence may have at least 80%, at least85%, at least 90%, at least 95%, at least 98% identity between two ormore of the HBV genotypes A-H.

In one embodiment, the target sequence may have identity between two ormore of HBV genotypes A, B, C and D. The target sequence may have atleast 80%, at least 85%, at least 90%, at least 95%, at least 98%between HBV genotypes A and B.

In one embodiment, the target sequence may have identity between two ormore of HBV genotypes A, B, C and D. The target sequence may have atleast 80%, at least 85%, at least 90%, at least 95%, at least 98%between HBV genotypes A and C.

In one embodiment, the target sequence may have identity between threeor more of HBV genotypes A, B, C and D. The target sequence may have atleast 80%, at least 85%, at least 90%, at least 95%, at least 98%identity between HBV genotypes A, B, C and D.

In one embodiment, the target sequence may have identity between threeor more of HBV genotypes A, B, C and D. The target sequence may have atleast 80%, at least 85%, at least 90%, at least 95%, at least 98%identity between HBV genotypes A, B and C.

In one embodiment, the target sequence may have identity between threeor more of HBV genotypes A, B, C and D. The target sequence may have atleast 80%, at least 85%, at least 90%, at least 95%, at least 98%identity between HBV genotypes A, B, and D.

In one embodiment, the target sequence may have identity between all ofHBV genotypes A, B, C and D. The target sequence may have at least 80%,at least 85%, at least 90%, at least 95%, at least 98% or at least 99%identity between HBV genotypes A, B, C and D.

In various embodiments, the target sequence is within the sequence shownas SEQ ID No. 3.

In one embodiment, the target sequence comprises at least part of a geneor a mRNA encoding HBx or HBsAg or a naturally-occurring variantthereof.

In various embodiments, the target sequence is HBx or HBsAg or anaturally-occurring variant thereof.

In various embodiments, the target sequence is within the sequence shownas SEQ ID No. 1.

In various embodiments, the target sequence is within the sequence shownas SEQ ID No. 2.

In various embodiments, the target sequence is selected from one or moreof the following positions in SEQ ID NO 3: position 1 to 1944, position157 to 1840, position 1196 to 1941, position 1376 to 1840 and position3158-3182. Preferably, the target sequence is selected from position1530 to 1598 of SEQ ID NO: 3, more preferable from position 1577 to 1598of SEQ ID NO: 3 and most preferably from position 1530 to 1543 of SEQ IDNO: 3.

In various embodiments, the target sequence may be selected from thegroup consisting of any one or more of positions:

-   -   1264-1278;    -   1265-1277;    -   1530-1543;    -   1530-1544;    -   1531-1543;    -   1551-1565;    -   1551-1566;    -   1577-1589;    -   1577-1591;    -   1577-1592;    -   1577 to 1598;    -   1578-1590;    -   1578-1592;    -   1583-1598;    -   1584-1598;    -   1585-1598;    -   670-706    -   670-684    -   691-705;    -   691-706;    -   692-706;    -   693-706;    -   694-706;    -   of SEQ ID No. 3.

In an aspect, the oligomer or oligomer conjugate of the invention iscapable of targeting from 8-30, 8-20, 8-18, 8-16, 8-14, 8-12 or 8-10contiguous nucleotides within the sequence shown as position 1200 to1900, preferably position 1530 to 1598, more preferable position 1577 to1598, most preferably position 1530 to 1543 of SEQ ID NO: 3 of SEQ IDNo. 3; preferably wherein said oligomer or oligomer conjugate iscomplementary to said contiguous nucleotides.

In an aspect, the oligomer or oligomer conjugate of the invention iscapable of targeting at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29 contiguous nucleotideswithin the sequence shown as position 1200 to 1900, preferably position1530 to 1598, more preferable position 1577 to 1598, most preferablyposition 1530 to 1543 of SEQ ID NO: 3 of SEQ ID No. 3; preferablywherein said oligomer or oligomer conjugate is complementary to saidcontiguous nucleotides.

In an aspect the oligomer or oligomer conjugate of the invention iscapable of targeting at least 8 contiguous nucleotides within thesequence shown as position 1200 to 1900, preferably position 1530 to1598, more preferable position 1577 to 1598, most preferably position1530 to 1543 of SEQ ID NO: 3 of SEQ ID NO: 3; preferably wherein saidoligomer or oligomer conjugate is complementary to said contiguousnucleotides.

In an aspect the oligomer or oligomer conjugate of the invention iscapable of targeting at least 9 contiguous nucleotides within thesequence shown as position 1200 to 1900, preferably position 1530 to1598, more preferable position 1577 to 1598, most preferably position1530 to 1543 of SEQ ID NO: 3 of SEQ ID NO: 3 preferably wherein saidoligomer or oligomer conjugate is complementary to said contiguousnucleotides.

In an aspect the oligomer or oligomer conjugate of the invention iscapable of targeting at least 10 contiguous nucleotides within thesequence shown as position 1200 to 1900, preferably position 1530 to1598, more preferable position 1577 to 1598, most preferably position1530 to 1543 of SEQ ID NO: 3 of SEQ ID NO: 3; preferably wherein saidoligomer or oligomer conjugate is complementary to said contiguousnucleotides.

In an aspect the oligomer or oligomer conjugate of the invention iscapable of targeting at least 11 contiguous nucleotides within thesequence shown as position 1200 to 1900, preferably position 1530 to1598, more preferable position 1577 to 1598, most preferably position1530 to 1543 of SEQ ID NO: 3 of SEQ ID NO: 3 preferably wherein saidoligomer or oligomer conjugate is complementary to said contiguousnucleotides.

In an aspect the oligomer or oligomer conjugate of the invention iscapable of targeting at least 12 contiguous nucleotides within thesequence shown as position 1200 to 1900, preferably position 1530 to1598, more preferable position 1577 to 1598, most preferably position1530 to 1543 of SEQ ID NO: 3 of SEQ ID NO: 3 preferably wherein saidoligomer or oligomer conjugate is complementary to said contiguousnucleotides.

In an aspect the oligomer or oligomer conjugate of the invention iscapable of targeting at least 13 contiguous nucleotides within thesequence shown as position 1200 to 1900, preferably position 1530 to1598, more preferable position 1577 to 1598, most preferably position1530 to 1543 of SEQ ID NO: 3 of SEQ ID NO: 3 preferably wherein saidoligomer or oligomer conjugate is complementary to said contiguousnucleotides.

In an aspect the oligomer or oligomer conjugate of the invention iscapable of targeting at least 14 contiguous nucleotides within thesequence shown as position 1200 to 1900, preferably position 1530 to1598, more preferable position 1577 to 1598, most preferably position1530 to 1543 of SEQ ID NO: 3 of SEQ ID NO: 3 preferably wherein saidoligomer or oligomer conjugate is complementary to said contiguousnucleotides.

In an aspect the oligomer or oligomer conjugate of the invention iscapable of targeting at least 15 contiguous nucleotides within thesequence shown as position 1200 to 1900, preferably position 1530 to1598, more preferable position 1577 to 1598, most preferably position1530 to 1543 of SEQ ID NO: 3 of SEQ ID NO: 3 preferably wherein saidoligomer or oligomer conjugate is complementary to said contiguousnucleotides.

In an aspect the oligomer or oligomer conjugate of the invention iscapable of targeting at least 16 contiguous nucleotides within thesequence shown as position 1200 to 1900, preferably position 1530 to1598, more preferable position 1577 to 1598, most preferably position1530 to 1543 of SEQ ID NO: 3 of SEQ ID NO: 3 preferably wherein saidoligomer or oligomer conjugate is complementary to said contiguousnucleotides.

In one embodiment, the target sequence comprises a sequence within thesequence set forth in SEQ ID NO: 1 or SEQ ID No 2 or SEQ ID No 3 or asequence having at least 80% identity thereto. Thus, the oligomer oroligomer conjugate can comprise or consist of a core motif selected fromthe group presented herein, wherein said oligomer or oligomer conjugate(or contiguous nucleotide portion thereof) may optionally have one, two,or three mismatches against said selected motif sequence:

(SEQ ID NO: 13) GCGTAAAGAGAGG; (SEQ ID NO: 20) AGCGAAGTGCACACG;(SEQ ID NO: 11) GCGTAAAGAGAGGT; (SEQ ID NO: 26) AGGTGAAGCGAAGTG;(SEQ ID NO 18) AGCGAAGTGCACACGG; (SEQ ID NO: 7) CGAACCACTGAACA;(SEQ ID NO 4) GAACCACTGAACAAA; (SEQ ID NO 5) CGAACCACTGAACAAA;(SEQ ID NO 6) CGAACCACTGAACAA; (SEQ ID NO 8) CGAACCACTGAAC (SEQ ID NO 9)CCGCAGTATGGATCG (SEQ ID NO: 10) CGCAGTATGGATC; (SEQ ID NO 12)CGCGTAAAGAGAGGT; (SEQ ID NO 14) AGAAGGCACAGACGG; (SEQ ID NO 15)GAGAAGGCACAGACGG (SEQ ID NO 16) GAAGTGCACACGG; (SEQ ID NO 17)GCGAAGTGCACACGG; (SEQ ID NO 19) CGAAGTGCACACG; (SEQ ID NO 27)AGGTGAAGCGAAGT; and (SEQ ID NO: 852) TAGTAAACTGAGCCA

In one embodiment, the target sequence comprises the sequence set forthbelow or a sequence having at least 80% identity to any thereto. Thus,the oligomer or oligomer conjugate can comprise or consist of a sequencehybridizing to a target sequence selected from the group presentedbelow, wherein said oligomer or oligomer conjugate (or contiguousnucleotide portion thereof) may optionally have one, two, or threemismatches against said selected target sequence.

Target Sequence acggggcgcacctctctttacgcg (SEQ ID NO: 827)cgtgtgcacttcgcttcacctc (SEQ ID NO: 828)ccgtctgtgccttctc (SEQ ID NO: 829) cgatccatactgcgg (SEQ ID NO: 830)tggctcagtttacta (SEQ ID NO: 831) ctagtgccatttgtt(SEQ ID NO: 833)

In some embodiments, the oligomer or oligomer conjugate may tolerate 1,2, 3, or 4 (or more) mismatches, when hybridising to the target sequenceand still sufficiently bind to the target to show the desired effect,i.e. down-regulation of the target. Mismatches may, for example, becompensated by increased length of the oligomer nucleotide sequenceand/or an increased number of nucleotide analogues, such as LNA, presentwithin the nucleotide sequence.

In some embodiments, the contiguous nucleotide sequence comprises nomore than 3, such as no more than 2 mismatches when hybridizing to thetarget sequence, such as to the corresponding region of a nucleic acidwhich encodes HBx or HBsAg.

In some embodiments, the contiguous nucleotide sequence comprises nomore than a single mismatch when hybridizing to the target sequence,such as the corresponding region of a nucleic acid which encodes HBx orHBsAg.

If the target is HBx, the nucleotide sequence of the oligomer oroligomer conjugate of the invention or the contiguous nucleotidesequence preferably has at least 80% identity (sometimes referred to ashomology or homologous) to a corresponding sequence selected from thenucleotide sequences presented herein, such as at least 85%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96% homologous, at least 97% homologous, at least 98%homologous, at least 99% homologous, such as 100% homologous(identical).

If the target is HBx, the nucleotide sequence of the oligomer oroligomer conjugate of the invention or the contiguous nucleotidesequence preferably has at least 80% identity (sometimes referred to ashomology or homologous) to the reverse complement of a correspondingsequence within the sequence presented as SEQ ID No. 1, such as at least85%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96% homologous, at least 97% homologous, atleast 98% homologous, at least 99% homologous, such as 100% homologous(identical).

If the target is HBx, the nucleotide sequence of the oligomer oroligomer conjugate of the invention or the contiguous nucleotidesequence is preferably at least 80% complementary to a sub-sequencepresent within the sequence presented as SEQ ID No. 1, such as at least85%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96% complementary, at least 97%complementary, at least 98% complementary, at least 99% complementary,such as 100% complementary (perfectly complementary).

In some embodiments if the target is HBx, the oligomer or oligomerconjugate (or contiguous nucleotide portion thereof) is selected from,or comprises, one of the sequences presented herein, or a sub-sequenceof at least 10 contiguous nucleotides thereof, wherein said oligomer oroligomer conjugate (or contiguous nucleotide portion thereof) mayoptionally comprise one, two, or three mismatches when compared to thesequence.

If the target is HBsAg, the nucleotide sequence of the oligomer oroligomer conjugate of the invention or the contiguous nucleotidesequence preferably has at least 80% identity (sometimes referred to ashomology or homologous) to a corresponding sequence selected from thegroup presented herein, such as at least 85%, at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96% homologous, at least 97% homologous, at least 98% homologous, atleast 99% homologous, such as 100% homologous (identical).

If the target is HBsAg, the nucleotide sequence of the oligomer oroligomer conjugate of the invention or the contiguous nucleotidesequence preferably has at least 80% identity (sometimes referred to ashomology or homologous) to the reverse complement of a correspondingsequence present within the sequence presented as SEQ ID No. 2, such asat least 85%, at least 90%, at least 91%, at least 92% at least 93%, atleast 94%, at least 95%, at least 96% homologous, at least 97%homologous, at least 98% homologous, at least 99% homologous, such as100% homologous (identical).

If the target is HBsAg, the nucleotide sequence of the oligomer oroligomer conjugate of the invention or the contiguous nucleotidesequence is preferably at least 80% complementary to a sub-sequencepresent within the sequence presented as SEQ ID No. 2, such as at least85%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96% complementary, at least 97%complementary, at least 98% complementary, at least 99% complementary,such as 100% complementary (perfectly complementary).

In some embodiments if the target is HBsAg, the oligomer or oligomerconjugate (or contiguous nucleotide portion thereof) is selected from,or comprises, one of the sequences selected from the group presentedherein, or a sub-sequence of at least 10 contiguous nucleotides thereof,wherein said oligomer or oligomer conjugate (or contiguous nucleotideportion thereof) may optionally comprise one, two, or three mismatcheswhen compared to the sequence.

In some embodiments the sub-sequence may consist of 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29 contiguousnucleotides, such as from 12-22, such as from 12-18, such as from 12-16nucleotides. Suitably, in some embodiments, the sub-sequence is of thesame length as the contiguous nucleotide sequence of the oligomer of theinvention.

However, it is recognised that, in some embodiments the nucleotidesequence of the oligomer or oligomer conjugate may comprise additional5′ or 3′ nucleotides, such as, independently, 1, 2, 3, 4 or 5 additionalnucleotides 5′ and/or 3′, which are non-complementary to the targetsequence. In this respect the oligomer of the invention, may, in someembodiments, comprise a contiguous nucleotide sequence which is flanked5′ and or 3′ by additional nucleotides. In some embodiments theadditional 5′ or 3′ nucleotides are naturally occurring nucleotides,such as DNA or RNA. In some embodiments, the additional 5′ or 3′nucleotides may represent region D as referred to in the context ofgapmer oligomer or oligomer conjugate herein.

RNAse Recruitment

It is recognised that an oligomeric molecule may function via non RNasemediated degradation of target mRNA, such as by steric hindrance oftranslation, or other methods.

For some embodiments, the oligomer or oligomer conjugate of theinvention is capable of recruiting an endoribonuclease (RNase), such asRNase H.

It is preferable that the oligomer or oligomer conjugate of theinvention comprises a region of at least 6, such as at least 7consecutive nucleotide units, such as at least 8 or at least 9consecutive nucleotide units (residues), including 7, 8, 9, 10, 11, 12,13, 14, 15 or 16 consecutive nucleotides, which, when formed in a duplexwith the complementary target RNA is capable of recruiting RNase. Thecontiguous sequence which is capable of recruiting RNAse may be region Xas referred to in the context of a gapmer as described herein. In someembodiments the size of the contiguous sequence which is capable ofrecruiting RNAse, such as region X, may be higher, such as 10, 11, 12,13, 14, 15, 16, 17, 18, 19 or 20 nucleotide units.

EP 1 222 309 provides in vitro methods for determining RNaseH activity,which may be used to determine the ability to recruit RNaseH. Anoligomer is deemed capable of recruiting RNase H if, when provided withthe complementary RNA target, it has an initial rate, as measured inpmol/l/min, of at least 1%, such as at least 5%, such as at least 10%or, more than 20% of the of the initial rate determined using DNA onlyoligonucleotide, having the same base sequence but containing only DNAunits, with no 2′ substitutions, with phosphorothioate linkage groupsbetween all units in the oligonucleotide, using the methodology providedby Examples 91-95 of EP 1 222 309.

In some embodiments, an oligomer is deemed essentially incapable ofrecruiting RNaseH if, when provided with the complementary RNA target,and RNaseH, the RNaseH initial rate, as measured in pmol/l/min, is lessthan 1%, such as less than 5%, such as less than 10% or less than 20% ofthe initial rate determined using the equivalent DNA onlyoligonucleotide, with no 2′ substitutions, with phosphorothioate linkagegroups between all nucleotides in the oligonucleotide, using themethodology provided by Examples 91-95 of EP 1 222 309.

In other embodiments, an oligomer is deemed capable of recruiting RNaseHif, when provided with the complementary RNA target, and RNaseH, theRNaseH initial rate, as measured in pmol/l/min, is at least 20%, such asat least 40%, such as at least 60%, such as at least 80% of the initialrate determined using the equivalent DNA only oligonucleotide, with no2′ substitutions, with phosphorothioate linkage groups between allnucleotides in the oligonucleotide, using the methodology provided byExamples 91-95 of EP 1 222 309.

Typically the region of the oligomer or oligomer conjugate of theinvention which forms the consecutive nucleotide units which, whenformed in a duplex with the complementary target RNA is capable ofrecruiting RNase consists of nucleotide units which form a DNA/RNA likeduplex with the RNA target—and include both DNA units and LNA unitswhich are in the alpha-L configuration, particularly preferred beingalpha-L-oxy LNA.

The oligomer or oligomer conjugate of the invention may comprise anucleotide sequence which comprises both nucleotides and nucleotideanalogues, and may be in the form of a gapmer, a headmer or a mixmer.

In some embodiments, in addition to enhancing affinity of the oligomerfor the target region, some nucleoside analogues also mediate RNase(e.g., RNaseH) binding and cleavage. Since α-L-LNA units recruit RNaseHactivity to a certain extent, in some embodiments, gap regions (e.g.,region X as referred to herein) of oligomers containing α-L-LNA unitsconsist of fewer units recognizable and cleavable by the RNaseH, andmore flexibility in the mixmer construction is introduced.

Synthesis

The present invention provides a method of manufacturing an oligomerconjugate, comprising conjugating at least one oligomer to a carriercomponent, wherein said oligomer conjugate is suitable for treating aviral disorder.

The present invention also provides a method of manufacturing apolyoligomer conjugate as described herein, comprising attaching one ormore oligomers to a linker group (E or L) or a symmetrical brancherregion F which is then attached to a carrier component as describedherein, wherein said oligomer conjugate is suitable for treating a viraldisorder.

In some embodiments the symmetrical brancher region is either1,3-pentylamidopropyl (from1,3-bis-[5-(4,4′-dimethoxytrityloxy)pentylamido]propyl-2-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite),tris-2,2,2-(propyloxymethyl)ethyl (fromtris-2,2,2-[3-(4,4′-dimethoxytrityloxy)propyloxymethyl]ethyl-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite)or tris-2,2,2-(propyloxymethyl)methyleneoxypropyl (fromtris-2,2,2-[3-(4,4′-dimethoxytrityloxy)propyloxymethyl]methyleneoxypropyl-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite).In some embodiments the asymmetrical brancher region is1,3-pentylamidopropyl (from1-[5-(4,4′-dimethoxytrityloxy)pentylamido]-3-[5-fluorenomethoxycarbonyloxypentylamido]-propyl-2-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite),Glen Reseach, USA provides such suitable branchers (e.g. catalog number0-1920, 10-1922 and 10-1925).

Linker groups and brancher regions as described herein may be cleavableor non-cleavable.

In a further aspect a method is provided for manufacturing thecomposition of the invention, comprising mixing the oligomer conjugateof the invention with a pharmaceutically acceptable diluent, solvent,carrier, salt and/or adjuvant.

Activated Oligomers

The term “activated oligomer,” as used herein, refers to an oligomer ofthe invention that is covalently linked (i.e., functionalized) to atleast one functional moiety that permits covalent linkage of theoligomer to one or more conjugated moieties, i.e., moieties that are notthemselves nucleic acids or units, to form the conjugates hereindescribed. Typically, a functional moiety will comprise a chemical groupthat is capable of covalently bonding to the oligomer via, e.g., a3′-hydroxyl group or the exocyclic NH₂ group of the adenine base, aspacer that is preferably hydrophilic and a terminal group that iscapable of binding to a conjugated moiety (e.g., an amino, sulfhydryl orhydroxyl group). In some embodiments, this terminal group is notprotected, e.g., is an NH₂ group. In other embodiments, the terminalgroup is protected, for example, by any suitable protecting group suchas those described in “Protective Groups in Organic Synthesis” byTheodora W Greene and Peter G M Wuts, 3rd edition (John Wiley & Sons,1999). Examples of suitable hydroxyl protecting groups include esterssuch as acetate ester, aralkyl groups such as benzyl, diphenylmethyl, ortriphenylmethyl, and tetrahydropyranyl. Examples of suitable aminoprotecting groups include benzyl, alpha-methylbenzyl, diphenylmethyl,triphenylmethyl, benzyloxycarbonyl, tert-butoxycarbonyl, and acyl groupssuch as trichloroacetyl or trifluoroacetyl. In some embodiments, thefunctional moiety is self-cleaving. In other embodiments, the functionalmoiety is biodegradable. See e.g., U.S. Pat. No. 7,087,229, which isincorporated by reference herein in its entirety.

In some embodiments, oligomers of the invention are functionalized atthe 5′ end in order to allow covalent attachment of the conjugatedmoiety to the 5′ end of the oligomer. In other embodiments, oligomers ofthe invention can be functionalized at the 3′ end. In still otherembodiments, oligomers of the invention can be functionalized along thebackbone or on the heterocyclic base moiety. In yet other embodiments,oligomers of the invention can be functionalized at more than oneposition independently selected from the 5′ end, the 3′ end, thebackbone and the base.

In some embodiments, activated oligomers of the invention aresynthesized by incorporating during the synthesis one or more units thatis covalently attached to a functional moiety. In other embodiments,activated oligomers of the invention are synthesized with units thathave not been functionalized, and the oligomer is functionalized uponcompletion of synthesis. In some embodiments, the oligomers arefunctionalized with a hindered ester containing an aminoalkyl linker,wherein the alkyl portion has the formula (CH₂)_(w), wherein w is aninteger ranging from 1 to 10, preferably about 6, wherein the alkylportion of the alkylamino group can be straight chain or branched chain,and wherein the functional group is attached to the oligomer via anester group (—O—C(O)—(CH₂)_(w)NH).

In other embodiments, the oligomers are functionalized with a hinderedester containing a (CH₂)_(w)-sulfhydryl (SH) linker, wherein w is aninteger ranging from 1 to 10, preferably about 6, wherein the alkylportion of the alkylamino group can be straight chain or branched chain,and wherein the functional group attached to the oligomer via an estergroup (—O—C(O)—(CH₂)_(w)SH). In some embodiments, sulfhydryl-activatedoligonucleotides are conjugated with polymer moieties such aspolyethylene glycol or peptides (via formation of a disulfide bond).

Activated oligomers containing hindered esters as described above can besynthesized by any method known in the art, and in particular by methodsdisclosed in PCT Publication No. WO 2008/034122 and the examplestherein, which is incorporated herein by reference in its entirety.

In still other embodiments, the oligomers of the invention arefunctionalized by introducing sulfhydryl, amino or hydroxyl groups intothe oligomer by means of a functionalizing reagent substantially asdescribed in U.S. Pat. Nos. 4,962,029 and 4,914,210, i.e., asubstantially linear reagent having a phosphoramidite at one end linkedthrough a hydrophilic spacer chain to the opposing end which comprises aprotected or unprotected sulfhydryl, amino or hydroxyl group. Suchreagents primarily react with hydroxyl groups of the oligomer. In someembodiments, such activated oligomers have a functionalizing reagentcoupled to a 5′-hydroxyl group of the oligomer. In other embodiments,the activated oligomers have a functionalizing reagent coupled to a3′-hydroxyl group. In still other embodiments, the activated oligomersof the invention have a functionalizing reagent coupled to a hydroxylgroup on the backbone of the oligomer. In yet further embodiments, theoligomer of the invention is functionalized with more than one of thefunctionalizing reagents as described in U.S. Pat. Nos. 4,962,029 and4,914,210, incorporated herein by reference in their entirety. Methodsof synthesizing such functionalizing reagents and incorporating theminto units or oligomers are disclosed in U.S. Pat. Nos. 4,962,029 and4,914,210.

In some embodiments, the 5′-terminus of a solid-phase bound oligomer isfunctionalized with a dienyl phosphoramidite derivative, followed byconjugation of the deprotected oligomer with, e.g., an amino acid orpeptide via a Diels-Alder cycloaddition reaction.

In various embodiments, the incorporation of units containing 2′-sugarmodifications, such as a 2′-carbamate substituted sugar or a2′-(O-pentyl-N-phthalimido)-deoxyribose sugar into the oligomerfacilitates covalent attachment of conjugated moieties to the sugars ofthe oligomer. In other embodiments, an oligomer with an amino-containinglinker at the 2′-position of one or more units is prepared using areagent such as, for example,5′-dimethoxytrityl-2′-O-(e-phthalimidylaminopentyl)-2′-deoxyadenosine-3-N,N-diisopropyl-cyanoethoxyphosphoramidite (see, e.g., Manoharan, et al., Tetrahedron Letters,1991, 34, 7171.)

In still further embodiments, the oligomers of the invention may haveamine-containing functional moieties on the nucleobase, including on theN6 purine amino groups, on the exocyclic N2 of guanine, or on the N4 or5 positions of cytosine. In various embodiments, such functionalizationmay be achieved by using a commercial reagent that is alreadyfunctionalized in the oligomer synthesis.

Some functional moieties are commercially available, for example,heterobifunctional and homobifunctional linking moieties are availablefrom the Pierce Co. (Rockford, Ill.). Other commercially availablelinking groups are 5-Amino-Modifier C6 and 3-Amino-Modifier reagents,both available from Glen Research Corporation (Sterling, Va.).5-Amino-Modifier C6 is also available from ABI (Applied Biosystems Inc.,Foster City, Calif.) as Aminolink-2, and 3-Amino-Modifier is alsoavailable from Clontech Laboratories Inc. (Palo Alto, Calif.).

Polyoligomers

As indicated above, in some embodiments of the present invention, theoligomer or the oligomer component of the oligomer conjugate maycomprise or be part of a molecule that has two targeting sequences. Insome instances, these molecules are called polyoligomers.

In some embodiments, the oligomer conjugate has or comprises thestructure:

-   -   Carrier component-L1-First Oligomer Region-L2-Second Oligomer        Region        -   wherein L1 is an optional linker or brancher region or            tether molecule or bridging moiety        -   wherein L2 is an optional linker or brancher region or            tether molecule or bridging moiety        -   wherein L1 and L2 can be the same or different; or            wherein said oligomer conjugate has or comprises the            structure:    -   First Oligomer Region-L2-Second Oligomer Region-L3-Carrier        component        -   wherein L2 is an optional linker or brancher region or            tether molecule or bridging moiety        -   wherein L3 is an optional linker or brancher region or            tether molecule or bridging moiety        -   wherein L2 and L3 can be the same or different different; or            wherein said oligomer conjugate has or comprises the            structure:    -   Carrier component 1-L1-First Oligomer Region-L2-Second Oligomer        Region-L3-Carrier component 2        -   wherein L1 is an optional linker or brancher region or            tether molecule or bridging moiety        -   wherein L2 is an optional linker or brancher region or            tether molecule or bridging moiety        -   wherein L3 is an optional linker or brancher region or            tether molecule or bridging moiety        -   wherein L1, L2 and L3 can be the same or different        -   wherein Carrier component 1 and Carrier component 2 can be            the same or different; or            wherein said oligomer conjugate has or comprises the            structure:    -   First Oligomer Region-L1-Carrier component 1-L2-Second Oligomer        Region        -   wherein L1 is an optional linker or brancher region or            tether molecule or bridging moiety        -   wherein L2 is an optional linker or brancher region or            tether molecule or bridging moiety        -   wherein L1 and L2 and L3 can be the same or different; or            wherein said oligomer conjugate has or comprises the            structure:    -   First Oligomer Region-L1-Carrier component 1-L2-Second Oligomer        Region-L3-Carrier component 2        -   wherein L1 is an optional linker or brancher region or            tether molecule or bridging moiety        -   wherein L2 is an optional linker or brancher region or            tether molecule or bridging moiety        -   wherein L3 is an optional linker or brancher region or            tether molecule or bridging moiety        -   wherein L1 and L2 can be the same or different        -   wherein Carrier component 1 and Carrier component 2 can be            the same or different; or            wherein said oligomer conjugate has or comprises the            structure:    -   Carrier component 1-L1-First Oligomer Region-L2-Carrier        component 2-L3-Second Oligomer Region-L4-Carrier component 3        -   wherein L1 is an optional linker or brancher region or            tether molecule or bridging moiety        -   wherein L2 is an optional linker or brancher region or            tether molecule or bridging moiety        -   wherein L3 is an optional linker or brancher region or            tether molecule or bridging moiety        -   wherein L1, L2 and L3 can be the same or different        -   wherein Carrier component 1, Carrier Component 2 and Carrier            component 3 can be the same or different.

In some embodiments, preferably the oligomer conjugate for the useaccording to the present invention wherein said oligomer conjugate hasor comprises the structure:

-   -   Carrier component-L1-First Oligomer Region-L2-Second Oligomer        Region        -   wherein L1 is an optional linker or brancher region or            tether molecule or bridging moiety        -   wherein L2 is an optional linker or brancher region or            tether molecule or bridging moiety        -   wherein L1 and L2 can be the same or different.

In some embodiments, the Linker 1 is present.

In some embodiments, the Linker 2 is present.

In some embodiments, the Linker 3 is present.

In some embodiments, the carrier component is linked, preferablyconjugated, to said first oligomer region.

In some embodiments, the carrier component is linked, preferablyconjugated, to the 5′ end of said oligomer.

In some embodiments, each of the first oligomer region and the secondoligomer regions is linked, preferably conjugated, by means of a linkeror brancher region.

In some embodiments, each of the first oligomer region and the secondoligomer regions is linked, preferably conjugated, by means of aphysiologically labile linker group or a physiologically labile brancherregion.

In some embodiments, the invention provides for a poly-oligomericcompound which may comprise a first region (region PA), a second region(region PB) and a third region (region PC), wherein the first region iscovalently linked to at least one further oligomeric compound (regionPA′), wherein the first region (region PA) and region PA′ are covalentlylinked via a biocleavable linker (region PB′), which may be, by way ofexample, as according to the second region as disclosed here, forexample a region of at least one phosphodiester linked DNA or RNA (suchas DNA), such as two, three, four or five phosphodiester linked DNA orRNA nucleosides (such as DNA nucleosides). Regions PB and PB′ may, insome embodiments have the same structure, e.g. the same number ofDNA/RNA nucleosides and phosphodiester linkages and/or the samenucleobase sequence. In other embodiments Regions PB and PB′ may bedifferent. By way of example such poly oligomeric compounds may have astructure such as: (5′-3′ or 3′-5′) Conjugate/CarrierCompound-PO—ON—PO′—ON′, wherein conjugate/carrier compound is region PC,PO is region PB, PO′ is region PB′, and ON is region PA, and ON′ isregion PA′.

It should be understood that region PA′ may, in some embodiments,comprise multiple further oligomeric compounds (such as a further 2 or 3oligomeric compounds) linked in series (or in parallel) via biocleavablelinkers, for example: Conjugate/Carrier Compound-PO—ON—PO—ON′—PO″—ON″,or Conjugate/Carrier Compound-PO—ON—[PO—ON′]n, wherein n may, forexample be 1, 2 or 3, and each ON′ may be the same or different, and ifdifferent may have the same or different targets.

In an aspect, the present invention employs poly-oligomeric compounds(also referred herein as oligomer compounds) for use in modulating, suchas inhibiting a target nucleic acid in a cell, for example HBV HBx orHBsAg. The oligomer compound comprises at least two oligomer regions,e.g. (PA and PA′) and may comprise further oligomer regions (e.g. PA″).At least one oligomer region is an oligomer which is capable ofmodulating a target sequence in HBx or HBsAg of HBV, for example anoligomer as provided by the present invention. In certain embodiments,each of PA, PA′ (and PA″ if present) may be an oligomer which is capableof modulating a target sequence in HBx or HBsAg of HBV, for example anoligomer as provided by the present invention. PA and PA′ may becomplements to different positions in the target sequence.

In some embodiments, PA may be an oligomer which is capable ofmodulating a target sequence in HBx and PA′ (and/or PA″ if present) maybe oligomers which are capable of modulating a different targetsequence. In certain embodiments, PA′ (and/or PA″ if present) may becapable of modulating a target sequence in HBsAg of HBV. For example PA′(and/or PA″ if present) may be an oligomer capable of modulating atarget sequence in HBsAg of HBV, as described herein.

In some embodiments, PA′ may be an oligomer which is capable ofmodulating a target sequence in HBx and PA (and/or PA″ if present) maybe oligomers which are capable of modulating a different target sequencefrom HBV. In certain embodiments, PA (and/or PA″ if present) may becapable of modulating a target sequence in HBsAg of HBV. For example PA(and/or PA″ if present) may be an oligomer capable of modulating atarget sequence in HBsAg of HBV, as described herein.

Each oligomer region may be flanked by a bio-cleavable region (regionPB), which may, for example, be a further region of 1-10 contiguousnucleotides (region PB), which comprise at least one phosphodiesterlinkage. Other physiological labile nucleoside regions may be used.

In some embodiments, the oligomer compounds of the invention arecovalently linked to a conjugate group, a targeting group, a reactivegroup, an activation group, or a blocking group, optionally, via a shortregion comprising (e.g. 1-10) of phosphodiester linked DNA or RNAnucleoside(s). Examples of such groups are the carrier components andconjugate components mentioned herein.

In some embodiments, the compound of the invention does not comprise RNA(units). In some embodiments, the compound according to the inventionforms a single contiguous sequence), optionally linked to a functiongroup, such as a conjugate group, and is such a linear molecule or issynthesized as a linear molecule. The oligomeric compound may thereforebe single stranded molecule. In some embodiments, the oligomer does notcomprise short regions of, for example, at least 3, 4 or 5 contiguousnucleotides, which are complementary to equivalent regions within thesame oligomeric compound (i.e. duplexes). The oligomer, in someembodiments, may be not (essentially) double stranded. In someembodiments, the oligomer is essentially not double stranded, such as isnot a siRNA.

Oligomer regions PA, PA′ and if present PA″ are phosphorothioateoligomers, i.e. at least 70% of the internucleoside linkages within eacholigomer region PA, PA′ and if present PA″, are phosphorothioatelinkages, such as at least 80% or at least 90% or all of theinternucleoside linkages if present oligomer regions PA, PA′ and PA″ (ifpresent), are phosphorothioate.

In some embodiments, oligomer regions PA, PA′ and if present PA″ mayform a single contiguous oligonucleotide sequence. Regions PA, PA′ andPA″ are interspaced by regions PB, for example regions of 1, 2, 3, 4, or5 phosphodiester linked DNA nucleosides.

When region PB comprises only 1 nucleoside, at least one, or both of theinternucleoside linkages between the region PB nucleoside (e.g. a DNAnucleoside) may be phosphodiester linkages. When region PB comprisesonly 2 or more nucleosides, the internucleoside linkages between theregion PB nucleoside (e.g. the DNA nucleosides) may be phosphodiesterlinkages and/or may be another internucleoside linkage, such asphosphorothioate linkages.

The oligomers of the invention, such as PA, PA′ and if present PA″, donot form part of a siRNA complex. The oligomers of the invention, suchas PA, PA′ and if present PA″, are non-complementary, e.g. they do nothybridize to one another to form a region of more than 8 or in someembodiments more than 6 contiguous base pairs. In some embodiments,regions PA and PA″ do not hybridize to one another to form a region ofmore than 4 contiguous base pairs. Exemplary base pairs may be betweenA-T, G-C or A-U. In the case there are three oligomer regions, PA, PA′and PA″, the non-complementarity is between PA and PA′, and PA′ and PA″,as well as PA and PA″.

The oligomer regions PA, PA′ and if present PA″ are not in the form of aduplex with a (substantially) complementary oligonucleotide—e.g. is notan siRNA.

In some embodiments, oligomer regions PA, PA′ and PA″ share the samecontiguous nucleotide sequence. In some embodiments, oligomer regions PAand PA′ share the same contiguous nucleotide sequence. In this respectthe invention provides for a single compound which can be used todeliver multiple copies of an oligomer (i.e. with the same contiguousnucleobase sequence and optionally the same chemical modifications) tothe target tissue.

The oligomer regions (PA, PA′ and if present PA″) are linked via atleast one biocleavable region, referred to as region PB herein (andwhere there is more than one region PB, region PB′ and region PB″). Insome embodiments, region PB comprises 1-10 nucleosides which form aphysiologically labile region between oligomer regions, or between an(or each) oligomer region and a linking group. Regions of DNAphosphodiester nucleosides may be used, but other nucleotide regions maybe used if they are suitably physiologically labile.

In some embodiments, the internucleoside linkage between the oligomerregion (PA, PA′ or if present PA″) and (each) second region PB, is aphosphodiester linked to the first (or only) DNA or RNA nucleoside ofregion PB comprises at least one phosphodiester linked DNA or RNAnucleoside.

The region PB may, in some embodiments, comprise further DNA or RNAnucleosides which may be phosphodiester linked.

As explained herein, region PB may also be used to join a functionalgroup to the oligomeric region(s), optionally via a further linkagegroup (PY). The use of region PB as a cleavable linker to joinfunctional groups to oligomer is described in detail inPCT/EP2013/073858, which is hereby incorporated by reference.

In some embodiments a region PB is further covalently linked to a thirdregion which may, for example, be a conjugate, a targeting group areactive group, and/or a blocking group (PC). Group (PC) may be acarrier component as described herein.

In some aspects, the present invention is based upon the provision of aphysiologically labile region, the second region, linking the firstregion, e.g. an antisense oligonucleotide, and a conjugate or functionalgroup, e.g. carrier component. The physiologically labile region maycomprises at least one phosphodiester linked nucleoside, such as a DNAor RNA nucleoside, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10phosphodiester linked nucleosides, such as DNA or RNA. In someembodiments, the oligomeric compound comprises a cleavable(physiologically labile) linker. In this respect the cleavable linker ispreferably present in region PB (or in some embodiments, between regionPA and PB).

In some embodiments, one (or more or all) region PB may comprise orconsists of at least one DNA or RNA nucleosides linked to the firstregion via a phosphodiester linkage. In some aspects, theinternucleoside linkage between an oligomer region and second region isconsidered as part of region PB.

In some embodiments, a (or more or each) region PB comprises or consistsof at least between 1 and 10 linked nucleosides, such as 1, 2, 3, 4, 5,6, 7, 8, 9 or 10 linked DNA or RNA nucleotides. Whilst a region ofDNA/RNA phosphodiester is considered important in the provision of acleavable linker, it is possible that region PB also comprisessugar-modified nucleoside analogues, such as those referred to under thefirst region above. However in some embodiments, the nucleosides ofregion PB are (optionally independently) selected from the groupconsisting of DNA and RNA. In some embodiments, the nucleosides ofregion PB are (optionally independently) DNA. It will be recognized thatthe nucleosides of region PB may comprise naturally occurring ornon-naturally occurring nucleobases. Typically, region PB comprises atleast one phosphodiester linked DNA or RNA nucleoside (which may, insome embodiments. be the first nucleoside adjacent to an oligomer). Ifregion PB comprises other nucleosides, region PB may also comprise ofother nucleoside linkages other than phosphodiester, such as (optionallyindependently) phosphorothioate, phosphodithioate, boranophosphate ormethyl phosphonate. However, in other exemplified embodiments, all theinternucleoside linkages in region PB are phosphorothioate. In someembodiments, all the nucleosides of region PB comprise (optionallyindependently) either a 2′-OH ribose sugar (RNA) or a 2′-H sugar—i.e.RNA or DNA. Between 1-5, or 1-4, such as 2, 3, 4 phosphate(phosphodiester) linked DNA nucleosides have been shown to beparticularly useful in the compounds of the invention.

In some embodiments, the second region comprises or consists of at leastbetween 1 and 10 (e.g. phosphodiester) linked DNA or RNA nucleosides,such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 (e.g. phosphodiester) linked DNAor RNA nucleotides.

In some embodiments, region PB comprises no more than 3 or no more than4 consecutive DNA or RNA nucleosides (such as DNA nucleosides). As suchregion PB may be so short as it does not recruit RNAseH, an aspect whichmay be important in embodiments when region PB does not form a part of asingle contiguous nucleobase sequence which is complementary to thetarget. Shorter region PBs, e.g. of 1-4 nts in length may also bepreferable in some embodiments, as they are unlikely to be the target ofsequence specific restriction enzymes. As such it is possible to varythe susceptibility of the region PB to endonuclease cleavage, andthereby fine-tune the rate of activation of the active oligomer in vivo,or even intra-cellular. Suitably, if very rapid activation is required,longer region PBs may be employed and/or region Bs which comprise therecognition sites of (e.g. cell or tissue specific or differentiallyexpressed) restriction enzymes.

In some embodiments, a region PB may be conjugated to a functional group(PC), such as a conjugate, targeting reactive group, an activationgroup, or blocking group, optionally via a linker group (PY such asthose provided herein). Functional groups may also be joined to anoligomer region, or the compound of the invention via other means, e.g.via phosphate nucleoside linkage (e.g. phosphodiester, phosphorothioate,phosphodithioate, boranophosphate or methylphosphonate) or a triazolgroup. In some aspects, the linkage group is the same as the region PBbetween at least two of the oligomer regions, and as such may be aphosphodiester linkage.

In some embodiments the DNA or RNA nucleotides of an (or more or each)region PB are independently selected from DNA and RNA nucleotides. Insome embodiments the DNA or RNA nucleotides of an (or more or each)region PB are DNA nucleotides. In some embodiments the DNA or RNAnucleotides of an (or more or each) region PB are RNA nucleotides.

In the context of the second region, the term DNA and RNA nucleoside maycomprise a naturally occurring or non-naturally occurring base (alsoreferred to as a base analogue or modified base).

It will be recognized that, in some embodiments, an (or more or each)region PB may further comprise other nucleotides or nucleotideanalogues. In some embodiments, (or more or each) region PB comprisesonly DNA or RNA nucleosides. In some embodiments, an (or more or each)region PB comprises more than one nucleoside, the internucleosidelinkages in an or each region PB comprise phosphodiester linkages. Insome embodiments, when an (or more or each) region PB comprises morethan one nucleoside, all the internucleoside linkages in the secondregion comprise phosphodiester linkages.

In some embodiments, at least two consecutive nucleosides of an (or moreor each) region PB are DNA nucleosides (such as at least 3 or 4 or 5consecutive DNA nucleotides). In some embodiments the at least twoconsecutive nucleosides an (or more or each) region PB are RNAnucleosides (such as at least 3 or 4 or 5 consecutive RNA nucleotides).In some embodiments the at least two consecutive nucleosides of the (ormore or each) region PB are at least one DNA and at least one RNAnucleoside. The internucleoside linkage between a region PA and regionPB may be a phosphodiester linkage. In some embodiments, when region PBcomprises more than one nucleoside, at least one further internucleosidelinkage is phosphodiester—such as the linkage group(s) between the 2 (or3 or 4 or 5) nucleosides adjacent to a region PA.

A region PB may be flanked on at least one side (either 5′ or 3′) by thefirst region, e.g. an antisense oligonucleotide, and on the other side(either 3′ or 5′ respectfully, via a further oligomer region (PA′), or aconjugate moiety or similar group (e.g. a blocking moiety/group, atargeting moiety/group or therapeutic small molecule moiety), optionallyvia a linker group (i.e. between the second region and theconjugate/blocking group etc. moiety).

In some embodiments, region PB does not form a complementary sequencewhen the oligomer region (e.g. PA, PA′ and/or PA″) and PB is aligned tothe complementary target sequence.

In some embodiments, region PB does form a complementary sequence whenthe oligomer region (e.g. PA, PA′ and/or PA″) and PB is aligned to thecomplementary target sequence. In this respect region PA and PB togethermay form a single contiguous sequence which is complementary to thetarget sequence.

In some embodiments, the sequence of bases in region PB is selected toprovide an optimal endonuclease cleavage site, based upon thepredominant endonuclease cleavage enzymes present in the target tissueor cell or sub-cellular compartment. In this respect, by isolating cellextracts from target tissues and non-target tissues, endonucleasecleavage sequences for use in region PB may be selected based upon apreferential cleavage activity in the desired target cell (e.g.liver/hepatocytes) as compared to a non-target cell (e.g. kidney). Inthis respect, the potency of the compound for target down-regulation maybe optimized for the desired tissue/cell.

In some embodiments region PB comprises a dinucleotide of sequence AA,AT, AC, AG, TA, TT, TC, TG, CA, CT, CC, CG, GA, GT, GC, or GG, wherein Cmay be 5-mthylcytosine, and/or T may be replaced with U.

In some embodiments region PB comprises a trinucleotide of sequence AAA,AAT, AAC, AAG, ATA, ATT, ATC, ATG, ACA, ACT, ACC, ACG, AGA, AGT, AGC,AGG, TAA, TAT, TAC, TAG, TTA, TTT, TTC, TAG, TCA, TCT, TCC, TCG, TGA,TGT, TGC, TGG, CAA, CAT, CAC, CAG, CTA, CTG, CTC, CTT, CCA, CCT, CCC,CCG, CGA, CGT, CGC, CGG, GAA, GAT, GAC, CAG, GTA, GTT, GTC, GTG, GCA,GCT, GCC, GCG, GGA, GGT, GGC, and GGG wherein C may be 5-mthylcytosineand/or T may be replaced with U.

In some embodiments region PB comprises a trinucleotide of sequenceAAAX, AATX, AACX, AAGX, ATAX, ATTX, ATCX, ATGX, ACAX, ACTX, ACCX, ACGX,AGAX, AGTX, AGCX, AGGX, TAAX, TATX, TACX, TAGX, TTAX, TTTX, TTCX, TAGX,TCAX, TCTX, TCCX, TCGX, TGAX, TGTX, TGCX, TGGX, CAAX, CATX, CACX, CAGX,CTAX, CTGX, CTCX, CTTX, CCAX, CCTX, CCCX, CCGX, CGAX, CGTX, CGCX, CGGX,GAAX, GATX, GACX, CAGX, GTAX, GTTX, GTCX, GTGX, GCAX, GCTX, GCCX, GCGX,GGAX, GGTX, GGCX, and GGGX, wherein X may be selected from the groupconsisting of A, T, U, G, C and analogues thereof, wherein C may be5-mthylcytosine and/or T may be replaced with U. It will be recognizedthat when referring to (naturally occurring) nucleobases A, T, U, G, C,these may be substituted with nucleobase analogues which function as theequivalent natural nucleobase (e.g. base pair with the complementarynucleoside).

In some embodiments, the compound of the invention may comprise morethan one conjugate group (or more than one functional group PX—such as aconjugate, targeting, blocking or activated group or a reactive oractivation group), such as 2 or 3 such groups. In some embodiments,region PB is covalently linked, optionally via a [e.g. non-nucleotide]linker group), to at least one functional group, such as two or threefunctional groups. In some embodiments, the first region (PA) may becovalently linked (e.g. via internucleoside linkages, such asphosphodiester linkages), to two region PBs, for example, one 5′ and one3′ to the first region PA, wherein each region PB may be (optionallyindependently) selected from the region PB described herein.

Composition

Oligomers of the invention and oligomer conjugates of the invention maybe used in pharmaceutical formulations and compositions. Suitably, suchcompositions comprise a pharmaceutically acceptable diluent, carrier,salt or adjuvant. WO 2007/03109 provides suitable and preferredpharmaceutically acceptable diluent, carrier and adjuvants—which arehereby incorporated by reference. Suitable dosages, formulations,administration routes, compositions, dosage forms, combinations withother therapeutic agents, pro-drug formulations are also provided in WO2007/03109—which are also hereby incorporated by reference.

Pharmaceutical compositions of the invention may include apharmaceutically acceptable carrier that may contain a variety ofcomponents that provide a variety of functions, including regulation ofdrug concentration, regulation of solubility, chemical stabilization,regulation of viscosity, absorption enhancement, regulation of pH, andthe like.

The pharmaceutical carrier may comprise a suitable liquid vehicle orexcipient and an optional auxiliary additive or additives. The liquidvehicles and excipients are conventional and commercially available.Illustrative thereof are distilled water, physiological saline, aqueoussolutions of dextrose, and the like. For water soluble formulations, thepharmaceutical composition preferably includes a buffer such as aphosphate buffer, or other organic acid salt, preferably at a pH in therange of 6.5 to 8. For formulations containing weakly soluble antisensecompounds, micro-emulsions may be employed, for example by using anonionic surfactant such as polysorbate 80 in an amount of 0.04-0.05%(w/v), to increase solubility. Other components may includeantioxidants, such as ascorbic acid, hydrophilic polymers, such as,monosaccharides, disaccharides, and other carbohydrates includingcellulose or its derivatives, dextrins, chelating agents, such as EDTA,and like components well known to those in the pharmaceutical sciences,e.g., Remington's Pharmaceutical Science, latest edition (MackPublishing Company, Easton, Pa.).

Oligonucleotides of the invention include the pharmaceuticallyacceptable salts thereof, including those of alkaline earth salts, e.g.,sodium or magnesium, ammonium or NX₄ ⁺, wherein X is C₁-C₄ alkyl. Otherpharmaceutically acceptable salts include organic carboxylic acids suchas formic, acetic, lactic, tartaric, malic, isethionic, lactobionic, andsuccinic acids; organic sulfonic acids such as methanesulfonic,ethanesulfonic, toluenesulfonic acid and benzenesulfonic; and inorganicacids such as hydrochloric, sulfuric, phosphoric, and sulfamic acids.Pharmaceutically acceptable salts of a compound having a hydroxyl groupinclude the anion of such compound with a suitable cation such as Na⁺,NH₄ ⁺, or the like.

The patient should receive a sufficient daily dosage of oligonucleotideto achieve an effective yet safe intercellular concentrations ofcombined oligonucleotides. Those skilled in the art should be readilyable to derive appropriate dosages and schedules of administration tosuit the specific circumstance and needs of the patient.

The effectiveness of the treatment may be assessed by routine methods,which are used for determining whether or not remission has occurred.Such methods generally depend upon morphological, cytochemical,cytogenetic, immunologic and molecular analyses. In addition, remissioncan be assessed genetically by probing the level of expression of one ormore relevant genes. The reverse transcriptase polymerase chain reaction(RT-PCR) methodology can be used to detect even very low numbers of mRNAtranscript.

Oligonucleotide Delivery Techniques

Oligonucleotides and conjugates of the invention may be preferablyadministered to a subject orally or topically but may also beadministered intravenously by injection. The vehicle is designedaccordingly. Alternatively, the oligonucleotide may be administeredsubcutaneously via controlled release dosage forms or conventionalformulation for intravenous injection. A preferred method ofadministration of oligonucleotides comprises either topical, systemic orregional perfusion, as is appropriate. According to a method of regionalperfusion, the afferent and efferent vessels supplying the extremitycontaining the lesion are isolated and connected to a low-flow perfusionpump in continuity with an oxygenator and a heat exchanger. The iliacvessels may be used for perfusion of the lower extremity. The axillaryvessels are cannulated high in the axilla for upper extremity lesions.Oligonucleotide is added to the perfusion circuit, and the perfusion iscontinued for an appropriate time period, e.g., one hour. Perfusionrates of from about 100 to about 150 ml/minute may be employed for lowerextremity lesions, while half that rate should be employed for upperextremity lesions. Systemic heparinization may be used throughout theperfusion, and reversed after the perfusion is complete. This isolationperfusion technique permits administration of higher doses ofchemotherapeutic agent than would otherwise be tolerated upon infusioninto the arterial or venous systemic circulation.

In a particular embodiment, oligomers and conjugates of the inventionare administered systemically or formulated for systemic administration.

For systemic infusion, the oligonucleotides are preferably delivered viaa central venous catheter, which is connected to an appropriatecontinuous infusion device. Indwelling catheters provide long termaccess to the intravenous circulation for frequent administration ofdrugs over extended time periods. They are generally surgically insertedinto the external cephalic or internal jugular vein under general orlocal anesthesia. The subclavian vein is another common site ofcatheterization. The infuser pump may be external, or may form part ofan entirely implantable central venous system such as the INFUSAPORTsystem available from Infusaid Corp., Norwood, Mass. and the PORT-A-CATHsystem available from Pharmacia Laboratories, Piscataway, N.J. Thesedevices are implanted into a subcutaneous pocket under local anesthesia.A catheter, connected to the pump injection port, is threaded throughthe subclavian vein to the superior vena cava. The implant contains asupply of oligonucleotide in a reservoir which may be replenished asneeded by injection of additional drug from a hypodermic needle througha self-sealing diaphragm in the reservoir. Completely implantableinfusers are preferred, as they are generally well accepted by patientsbecause of the convenience, ease of maintenance and cosmetic advantageof such devices.

Oligonucleotides and conjugates of the invention may be introduced byany of the methods described in U.S. Pat. No. 4,740,463, incorporatedherein by reference. One technique is in vitro transfection, which canbe done by several different methods. One method of transfectioninvolves the addition of DEAE-dextran to increase the uptake of thenaked DNA molecules by a recipient cell. See McCutchin, J. H. andPagano, J. S., J. Natl. Cancer Inst. 41, 351-7 (1968). Another method oftransfection is the calcium phosphate precipitation technique whichdepends upon the addition of Ca²⁺ to a phosphate-containing DNAsolution. The resulting precipitate apparently includes DNA inassociation with calcium phosphate crystals. These crystals settle ontoa cell monolayer; the resulting apposition of crystals and cell surfaceappears to lead to uptake of the DNA. A small proportion of the DNAtaken up becomes expressed in a transfectant, as well as in its clonaldescendants. See Graham, F. L. and van der Eb, A. J., Virology 52,456-467 (1973) and Virology 54, 536-539 (1973).

Transfection may also be carried out by cationic phospholipid-mediateddelivery. In particular, polycationic liposomes can be formed fromN-[1-(2,3-di-oleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOT-MA).See Feigner et al., Proc. Natl. Acad. Sci., 84, 7413-7417 (1987)(DNA-transfection); Malone et al., Proc. Natl. Acad. Sci., 86, 6077-6081(1989) (RNA-transfection).

For systemic or regional in vivo administration, the amount ofoligonucleotides may vary depending on the nature and extent of thedisease, the particular oligonucleotides utilized, and other factors.The actual dosage administered may take into account the size and weightof the patient, whether the nature of the treatment is prophylactic ortherapeutic in nature, the age, health and sex of the patient, the routeof administration, whether the treatment is regional or systemic, andother factors.

In addition to administration with conventional pharmaceutical carriers,the antisense oligonucleotides may be administered by a variety ofspecialized oligonucleotide delivery techniques. Sustained releasesystems suitable for use with the pharmaceutical compositions of theinvention include semi-permeable polymer matrices in the form of films,microcapsules, or the like, which may comprise polylactides; copolymersof L-glutamic acid and gamma-ethyl-L-glutamate, poly(2-hydroxyethylmethacrylate), and like materials, e.g., Rosenberg et al., Internationalapplication PCT/US92/05305.

The oligonucleotides and conjugates may be encapsulated in liposomes fortherapeutic delivery, as described for example in Liposome Technology,Vol. II, Incorporation of Drugs, Proteins, and Genetic Material, CRCPress. The oligonucleotide, depending upon its solubility, may bepresent both in the aqueous layer and in the lipidic layer, or in whatis generally termed a liposomic suspension. The hydrophobic layer,generally but not exclusively, comprises phospholipids such as lecithinand sphingomyelin, steroids such as cholesterol, ionic surfactants suchas diacetylphosphate, stearylamine, or phosphatidic acid, and/or othermaterials of a hydrophobic nature. Also comprised are the novel cationicamphiphiles, termed “molecular umbrellas”, that are described in (DeLonget al, Nucl. Acid. Res., 1999, 27(16), 3334-3341).

The embodiments of the present invention may be delivered by means ofparticulate systems and/or polymers. Particulate systems and polymersfor in vitro and in vivo delivery of polynucleotides have beenextensively reviewed by Feigner in Advanced Drug Delivery Reviews 5,163-187 (1990). Techniques for direct delivery are also described inCook S. T. Antisense Drug Technology, Principles, Strategies, andApplications, Marcel Dekker, Inc, 2001.

Pro-Drugs

The oligonucleotides may be synthesized as pro-drugs carrying lipophilicgroups, such as for example methyl-SATE (S-acetylthioethyl) or t-Bu-SATE(S-pivaloylthioethyl) protecting groups, that confers nucleaseresistance to the oligo, improve cellular uptake and selectivelydeprotects after entry into the cell as described in Vives et al. Nucl.Acids Res. 1999, Vol. 27, 4071-4076.

Circular Molecules

The oligonucleotides may be synthesized as circular molecules in whichthe 5′ and 3′ ends of the oligonucleotides are covalently linked or heldtogether by an affinity pair one member of which is attached covalentlyto the 5′ end and the other attached covalently to the 3′end. Suchcircularization protects the oligonucleotide against degradation byexonucleases and may also improve cellular uptake and distribution. Inone aspect of the invention the moiety linking the 5′ and 3′ end of acircular oligonucleotide is cleaved automatically upon entry into anytype of human or vertebrate cell thereby linearising the oligonucleotideand enabling it to efficiently hybridize to its target sequence. Inanother aspect, the moiety linking the 5′ and 3′ends of theoligonucleotide is so designed that cleavage preferably occurs only inthe particular type of cells that expresses the mRNA that is the targetfor the antisense oligonucleotide. For instance, a circular antisenseoligonucleotide directed against a gene involved in a viral disorder maybe brought into action by linearisation only in the subset of cellsexpressing the gene in question, for example HBV HBx or HBsAg.

Additional Pharmaceutical Entity

Oligomers and oligomer conjugates of the invention may be used as theprimary therapeutic for the treatment of the disease state, or may beused in combination with non-oligonucleotide drugs.

Accordingly, the present invention provides a pharmaceutical systemcomprising a pharmaceutical composition as described herein and anadditional pharmaceutical entity. The additional pharmaceutical entitymay be any therapeutic agent known in the art. For example, theadditional pharmaceutical entity may be an antibody, a small moleculetherapeutic, a polynucleotide or gene therapy vector (e.g. a vectorcapable of expressing therapeutic polypeptides or RNAi agents).

Hence, the oligomer or the oligomer conjugate of the present inventionmay be used in combination with other actives, such as other anti-viralactives.

By way of example, the oligomer or the oligomer conjugate of the presentinvention may be used in combination with other actives, such asoligonucleotide-based antivirals—such as sequence specificoligonucleotide-based antivirals—acting either through antisense(including other LNA oligomers), siRNAs (such as ARC520), aptamers,morpholinos or any other antiviral, nucleotide sequence-dependent modeof action.

By way of further example, the oligomer or the oligomer conjugate of thepresent invention may be used in combination with other actives, such asimmune stimulatory antiviral compounds, such as interferon (e.g.pegylated interferon alpha), TLR7 agonists (e.g. GS-9620), ortherapeutic vaccines.

By way of further example, the oligomer or the oligomer conjugate of thepresent invention may be used in combination with other actives, such assmall molecules, with antiviral activity. These other actives could be,for example, nucleoside/nucleotide inhibitors (eg entecavir or tenofovirdisoproxil fumarate), encapsidation inhibitors, entry inhibitors (egMyrcludex B).

In certain embodiments, the additional therapeutic agent may be an HBVagent, an Hepatitis C virus (HCV) agent, a chemotherapeutic agent, anantibiotic, an analgesic, a nonsteroidal anti-inflammatory (NSAID)agent, an antifungal agent, an antiparasitic agent, an anti-nauseaagent, an anti-diarrheal agent, or an immunosuppressant agent.

In particular related embodiments, the additional HBV agent may beinterferon alpha-2b, interferon alpha-2a, and interferon alphacon-1(pegylated and unpegylated), ribavirin; an HBV RNA replicationinhibitor; a second antisense oligomer; an HBV therapeutic vaccine; anHBV prophylactic vaccine; lam ivudine (3TC); entecavir (ETV); tenofovirdiisoproxil fumarate (TDF); telbivudine (LdT); adefovir; or an HBVantibody therapy (monoclonal or polyclonal).

In other particular related embodiments, the additional HCV agent may beinterferon alpha-2b, interferon alpha-2a, and interferon alphacon-1(pegylated and unpegylated); ribavirin; an HCV RNA replication inhibitor(e.g., ViroPharma's VP50406 series); an HCV antisense agent; an HCVtherapeutic vaccine; an HCV protease inhibitor; an HCV helicaseinhibitor; or an HCV monoclonal or polyclonal antibody therapy.

The additional pharmaceutical entity may be an oligomer or oligomerconjugate as defined herein.

In certain embodiments the pharmaceutical system may comprise at leastone, at least two, at least three, up to a plurality of oligomers oroligomer conjugates provided by the present invention.

In certain embodiments the additional pharmaceutical entity may anoligomer or oligomer conjugate capable of modulating a target sequencein HBV. The oligomers or conjugates may each be capable of modulating atarget sequence in HBV HBx or HBsAg. The oligomers or conjugates may becapable of modulating a target sequence in HBV which is not within HBxor HBsAg. For example, at least one oligomer or conjugate may be capableof modulating a target sequence within the HBV gene or mRNA for HBcAg,HBeAg, or DNA polymerase.

In certain embodiments, the oligomer, or additional oligomer, orconjugate, or additional conjugate, may be capable of modulating atarget sequence in the HBV gene or mRNA for HBsAg.

When a combination of oligonucleotides targeting different targetsequences are employed, the ratio of the amounts of the different typesof oligonucleotide may vary over a broad range. According to onepreferred embodiment of the invention, the oligonucleotides of all typesare present in approximately equal amounts, by molarity.

Administration and Dosage

The present invention also relates pharmaceutical compositions thatcontain a therapeutically effective amount of a conjugate of theinvention. The composition can be formulated for use in a variety ofdrug delivery systems. One or more physiologically acceptable excipientsor carriers can also be included in the composition for properformulation. Suitable formulations for use in the present invention arefound in Remington's Pharmaceutical Sciences, Mack Publishing Company,Philadelphia, Pa., 17th ed., 1985. For a brief review of methods fordrug delivery, see, e.g., Langer (Science 249:1527-1533, 1990).

The pharmaceutical compositions of the present invention are intendedfor parenteral, intranasal, topical, oral, or local administration, suchas by a transdermal means, for prophylactic and/or therapeutictreatment. The pharmaceutical compositions can be administeredparenterally (e.g., by intravenous, intramuscular, or subcutaneousinjection), or by oral ingestion, or by topical application orintraarticular injection. Additional routes of administration includeintravascular, intra-arterial, intratumor, intraperitoneal,intraventricular, intraepidural, as well as nasal, ophthalmic,intrascleral, intraorbital, rectal, topical, or aerosol inhalationadministration. Sustained release administration is also specificallyincluded in the invention, by such means as depot injections or erodibleimplants or components. Thus, the invention provides compositions forparenteral administration that comprise the above mention agentsdissolved or suspended in an acceptable carrier, preferably an aqueouscarrier, e.g., water, buffered water, saline, PBS, and the like. Thecompositions may contain pharmaceutically acceptable auxiliarysubstances as required to approximate physiological conditions, such aspH adjusting and buffering agents, tonicity adjusting agents, wettingagents, detergents and the like. The invention also providescompositions for oral delivery, which may contain inert ingredients suchas binders or fillers for the formulation of a tablet, a capsule, andthe like. Furthermore, this invention provides compositions for localadministration, which may contain inert ingredients such as solvents oremulsifiers for the formulation of a cream, an ointment, and the like.

In a particular embodiment, oligomers and conjugates of the inventionare administered subcutaneously or formulated for subcutaneousadministration.

These compositions may be sterilized by conventional sterilizationtechniques, or may be sterile filtered. The resulting aqueous solutionsmay be packaged for use as is, or lyophilized, the lyophilizedpreparation being combined with a sterile aqueous carrier prior toadministration. The pH of the preparations typically will be between 3and 11, more preferably between 5 and 9 or between 6 and 8, and mostpreferably between 7 and 8, such as 7 to 7.5. The resulting compositionsin solid form may be packaged in multiple single dose units, eachcontaining a fixed amount of the above-mentioned agent or agents, suchas in a sealed package of tablets or capsules. The composition in solidform can also be packaged in a container for a flexible quantity, suchas in a squeezable tube designed for a topically applicable cream orointment.

The compositions containing an effective amount can be administered forprophylactic or therapeutic treatments. In prophylactic applications,compositions can be administered to a subject with a clinicallydetermined predisposition or increased susceptibility to development ofa tumor or cancer, neurodegenerative disease, or lysosomal disorder.Compositions of the invention can be administered to the patient (e.g.,a human) in an amount sufficient to delay, reduce, or preferably preventthe onset of clinical disease or tumorigenesis. In therapeuticapplications, compositions are administered to a subject (e.g., a human)already suffering from disease (e.g., a cancer, neurodegenerativedisease, or lysosomal storage disorder) in an amount sufficient to cureor at least partially arrest the symptoms of the condition and itscomplications. An amount adequate to accomplish this purpose is definedas a “therapeutically effective dose,” an amount of a compoundsufficient to substantially improve some symptom associated with adisease or a medical condition. For example, in the treatment of cancer,neurodegenerative disease, or lysosomal storage disease, an agent orcompound which decreases, prevents, delays, suppresses, or arrests anysymptom of the disease or condition would be therapeutically effective.A therapeutically effective amount of an agent or compound is notrequired to cure a disease or condition but will provide a treatment fora disease or condition such that the onset of the disease or conditionis delayed, hindered, or prevented, or the disease or condition symptomsare ameliorated, or the term of the disease or condition is changed or,for example, is less severe or recovery is accelerated in an individual.Amounts effective for this use may depend on the severity of the diseaseor condition and the weight and general state of the patient, butgenerally range from about 0.5 mg to about 3000 mg of the agent oragents per dose per patient. Suitable regimes for initial administrationand booster administrations are typified by an initial administrationfollowed by repeated doses at one or more hourly, daily, weekly, ormonthly intervals by a subsequent administration. The total effectiveamount of an agent present in the compositions of the invention can beadministered to a mammal as a single dose, either as a bolus or byinfusion over a relatively short period of time, or can be administeredusing a fractionated treatment protocol, in which multiple doses areadministered over a more prolonged period of time (e.g., a dose every4-6, 8-12, 14-16, or 18-24 hours, or every 2-4 days, 1-2 weeks, once amonth). Alternatively, continuous intravenous infusion sufficient tomaintain therapeutically effective concentrations in the blood arecontemplated.

The therapeutically effective amount of one or more agents presentwithin the compositions of the invention and used in the methods of thisinvention applied to mammals (e.g., humans) can be determined by theordinarily-skilled artisan with consideration of individual differencesin age, weight, and the condition of the mammal. The agents of theinvention are administered to a subject (e.g. a mammal, such as a human)in an effective amount, which is an amount that produces a desirableresult in a treated subject (e.g. the slowing or remission of a canceror neurodegenerative disorder). Therapeutically effective amounts can bedetermined empirically by those of skill in the art.

The patient may also receive an agent in the range of about 0.1 to 3,000mg per dose one or more times per week (e.g., 2, 3, 4, 5, 6, or 7 ormore times per week), 0.1 to 2,500 (e.g., 2,000, 1,500, 1,000, 500, 100,10, 1, 0.5, or 0.1) mg dose per week. A patient may also receive anagent of the composition in the range of 0.1 to 3,000 mg per dose onceevery two or three weeks. Single or multiple administrations of thecompositions of the invention comprising an effective amount can becarried out with dose levels and pattern being selected by the treatingphysician. The dose and administration schedule can be determined andadjusted based on the severity of the disease or condition in thepatient, which may be monitored throughout the course of treatmentaccording to the methods commonly practiced by clinicians or thosedescribed herein.

The carrier and conjugates of the present invention may be used incombination with either conventional methods of treatment or therapy ormay be used separately from conventional methods of treatment ortherapy.

When the conjugates of this invention are administered in combinationtherapies with other agents, they may be administered sequentially orconcurrently to an individual. Alternatively, pharmaceuticalcompositions according to the present invention may be comprised of acombination of a carrier-agent conjugate of the present invention inassociation with a pharmaceutically acceptable excipient, as describedherein, and another therapeutic or prophylactic agent known in the art.

Further Applications

The oligomers of the invention may be utilized as research reagents for,for example, diagnostics, therapeutics and prophylaxis.

In research, such oligomers may be used to specifically inhibit thesynthesis of the expression product of a target sequence (typically bydegrading or inhibiting the mRNA and thereby prevent protein formation)in cells and experimental animals thereby facilitating functionalanalysis of the target or an appraisal of its usefulness as a target fortherapeutic intervention.

In diagnostics the oligomers may be used to detect and quantitateexpression of the expression product of a target sequence in cell andtissues by northern blotting, in-situ hybridisation or similartechniques.

For therapeutics, an animal or a human, suspected of having a disease ordisorder, which can be treated by modulating the expression of a targetsequence is treated by administering oligomeric compounds in accordancewith this invention. Further provided are methods of treating a mammal,such as treating a human, suspected of having or being prone to adisease or condition, associated with expression of a target sequence byadministering a therapeutically or prophylactically effective amount ofone or more of the oligomers or compositions of the invention. Theoligomer, a conjugate or a pharmaceutical composition according to theinvention is typically administered in an effective amount.

The invention also provides for the use of the compound or conjugate ofthe invention as described for the manufacture of a medicament for thetreatment of a disorder as referred to herein, or for a method of thetreatment of as a disorder as referred to herein.

The invention also provides for a method for treating a disorder asreferred to herein said method comprising administering a compoundaccording to the invention as herein described, and/or a conjugateaccording to the invention, and/or a pharmaceutical compositionaccording to the invention to a patient in need thereof.

Viral Disorders

The oligomers, oligomer conjugates and other compositions according tothe invention can be used for the treatment of conditions associatedwith the target sequence, such as over-expression or expression of amutated version of the target sequence.

The invention further provides use of a compound of the invention in themanufacture of a medicament for the treatment of a disease, disorder orcondition as referred to herein.

In the context of the present invention, said disease, disorder orcondition may be a viral disorder.

In one embodiment, the viral disorder is associated with expression orover-expression of HBx or HBsAg. The viral disorder may be a disorderassociated with HBV. Examples of such viral disorders include, but arenot limited to, hepatitis B, cirrhosis, liver cancer (e.g.hepatocellular carcinoma), cholangiocarcinoma.

In one embodiment, the viral disorder is hepatitis B. As one of ordinaryskill would recognise, the term “hepatitis B” in the context of thepresent invention refers to an infectious disorder of the liver causedby HBV. Hepatitis B may be acute or chronic. Acute disease causes liverinflammation, vomiting, jaundice and occasionally death. Chronichepatitis B may cause cirrhosis and liver cancer.

In one embodiment, the viral disorder is cirrhosis. As one of ordinaryskill would recognise, the term “cirrhosis” in the context of thepresent invention refers to an advanced liver disease characterised bythe presence of fibrosis and regenerative nodules in the liver. Thesechanges can lead to a loss a liver function.

In one embodiment, the viral disorder in liver cancer. As one ofordinary skill would recognise, “liver cancer” in the context of thepresent invention refers to a malignancy which originates in the liver.

In one embodiment, the viral disorder is hepatocellular carcinoma (HCC).As one of ordinary skill would recognise, “HCC” in the context of thepresent invention refers to a type of liver cancer which commonly occurssecondary to a viral hepatitis infection, for example hepatitis B.Macroscopically, HCC appears as a nodular or infiltrative tumour. Thenodular type may be solitary (large mass) or multiple (when developed asa complication of cirrhosis). Tumour nodules are round to oval, grey orgreen (if the tumour produces bile), well circumscribed but notencapsulated. The diffuse type is poorly circumscribed and infiltratesthe portal veins, or the hepatic veins (rarely). Microscopically, thereare four architectural and cytological types (patterns) of HCC:fibrolamellar, pseudoglandular (adenoid), pleomorphic (giant cell) andclear cell. In well differentiated forms, tumour cells resemblehepatocytes, form trabeculae, cords and nests, and may contain bilepigment in cytoplasm. In poorly differentiated forms, malignantepithelial cells are discohesive, pleomorphic, anaplastic, giant. Thetumour has a scant stroma and central necrosis because of the poorvascularization.

In one embodiment, the viral disorder is cholangiocarcinoma. As one ofordinary skill would recognise, “cholangiocarcinoma” in the context ofthe present invention refers to a form of cancer that is composed ofmutated epithelial cells (or cells showing characteristics of epithelialdifferentiation) that originate in the bile ducts which drain bile fromthe liver into the small intestine.

Whilst various embodiments disclosed herein are related to a viraldisorder associated with HBV infection, the present invention is notlimited by these exemplary embodiments. Rather the present invention isapplicable to any disorder associated with a viral infection.

Generally stated, one aspect of the invention is directed to a method oftreating a mammal suffering from or susceptible to conditions associatedwith abnormal levels of the expression product of the target sequence,comprising administering to the mammal a therapeutically effectiveamount of an oligomer targeted to the target sequence. The oligomer ofthe invention may comprise one or more LNA units. The oligomer, aconjugate or a pharmaceutical composition according to the invention istypically administered in an effective amount.

An interesting aspect of the invention is directed to the use of anoligomer (compound) as defined herein or a oligomer conjugate as definedherein for the preparation of a medicament for the treatment of adisease, disorder or condition as referred to herein.

The methods of the invention are preferably employed for treatment orprophylaxis against diseases caused by abnormal levels of HBx or HBsAg.

Alternatively stated, in some embodiments, the invention is furthermoredirected to a method for treating abnormal levels of HBx or HBsAg, saidmethod comprising administering a oligomer of the invention, or aconjugate of the invention or a pharmaceutical composition of theinvention to a patient in need thereof.

The invention also relates to an oligomer, a composition or an oligomerconjugate as defined herein for use as a medicament.

The invention further relates to use of a compound, composition, or aconjugate as defined herein for the manufacture of a medicament for thetreatment of abnormal levels of HBx or HBsAg or expression of mutantforms of HBx or HBsAg (such as allelic variants, such as thoseassociated with one of the diseases referred to herein).

Moreover, the invention relates to a method of treating a subjectsuffering from a disease or condition such as those referred to herein.

A patient who is in need of treatment is a patient suffering from orlikely to suffer from the disease or disorder.

Example Oligomers

Examples of oligomers for use in the present invention are presented inthe following Tables.

TABLE 1 oligonucleotide sequence motifs used to design LNA modifiedoligomers. The table indicates the fraction of conservationwithin all published full length genotype sequences ofgenotype A, B, C and D (GenBank), meaning that a givenoligomer motif will be 100% complementary to the givenfraction of target sequences within a genotype. Alloligomer motifs were selected such that they willessentially target almost all sequences within genotypes A,B, C and D, thereby allowing for treatment of individualinfected with any of these four genotypes. Fraction of conservationStart within all position sequences of SEQ Target Target onOligosequence genotype ID No. HBx HBsAg U95551 motif A B C D SeqID 29 X 201 AAAACCCCGCCTGT 0.97 0.98 0.95 0.99 SeqID 30 X  202 AAAACCCCGCCTG0.96 0.97 0.97 0.97 SeqID 31 X  245 ACGAGTCTAGACTCT 0.96 0.97 0.97 0.97SeqID 32 X  245 CACGAGTCTAGACTCT 0.96 0.97 0.97 0.97 SeqID 33 X  246ACGAGTCTAGACTC 0.96 0.97 0.97 0.97 SeqID 34 X  246 CACGAGTCTAGACTC 0.960.97 0.97 0.97 SeqID 35 X  246 CCACGAGTCTAGACTC 0.96 0.97 0.97 0.97SeqID 36 X  247 ACGAGTCTAGACT 0.96 0.97 0.97 0.97 SeqID 37 X  247CACGAGTCTAGACT 0.96 0.97 0.97 0.97 SeqID 38 X  247 CCACGAGTCTAGACT 0.960.97 0.97 0.97 SeqID 39 X  247 ACCACGAGTCTAGACT 0.96 0.98 0.97 0.98SeqID 40 X  248 ACGAGTCTAGAC 0.96 0.97 0.97 0.98 SeqID 41 X  248CACGAGTCTAGAC 0.96 0.97 0.97 0.98 SeqID 42 X  248 CCACGAGTCTAGAC 0.960.97 0.97 0.97 SeqID 43 X  248 ACCACGAGTCTAGAC 0.96 0.97 0.96 0.97SeqID 44 X  248 CACCACGAGTCTAGAC 0.99 0.97 0.97 0.98 SeqID 45 X  249ACCACGAGTCTAGA 0.99 0.97 0.97 0.98 SeqID 46 X  249 CACCACGAGTCTAGA 0.990.97 0.97 0.98 SeqID 47 X  249 CCACCACGAGTCTAGA 0.99 0.98 0.97 0.98SeqID 48 X  250 CCACCACGAGTCTAG 0.99 0.98 0.97 0.98 SeqID 49 X  250TCCACCACGAGTCTAG 0.99 0.98 0.97 0.98 SeqID 50 X  251 CCACCACGAGTCTA 0.990.98 0.97 0.98 SeqID 51 X  251 TCCACCACGAGTCTA 0.98 0.97 0.97 0.98SeqID 52 X  251 GTCCACCACGAGTCTA 0.99 0.98 0.97 0.98 SeqID 53 X  252TCCACCACGAGTCT 0.98 0.97 0.97 0.98 SeqID 54 X  252 GTCCACCACGAGTCT 0.980.97 0.97 0.98 SeqID 55 X  252 AGTCCACCACGAGTCT 0.98 0.97 0.97 0.98SeqID 56 X  253 GTCCACCACGAGTC 0.98 0.97 0.97 0.98 SeqID 57 X  253AGTCCACCACGAGTC 0.98 0.97 0.97 0.98 SeqID 58 X  253 AAGTCCACCACGAGTC0.98 0.97 0.98 0.99 SeqID 59 X  254 AGTCCACCACGAGT 0.98 0.97 0.98 0.99SeqID 60 X  254 AAGTCCACCACGAGT 0.98 0.97 0.97 0.99 SeqID 61 X  254GAAGTCCACCACGAGT 0.98 0.97 0.98 0.99 SeqID 62 X  255 AAGTCCACCACGAG 0.980.97 0.98 0.99 SeqID 63 X  255 GAAGTCCACCACGAG 0.97 0.97 0.98 0.99SeqID 64 X  255 AGAAGTCCACCACGAG 0.97 0.97 0.98 0.99 SeqID 65 X  256AGAAGTCCACCACGA 0.97 0.97 0.98 0.99 SeqID 66 X  256 GAGAAGTCCACCACGA0.97 0.97 0.98 0.99 SeqID 67 X  257 GAGAAGTCCACCACG 0.97 0.97 0.97 0.98SeqID 68 X  257 AGAGAAGTCCACCACG 0.98 0.99 0.98 0.99 SeqID 69 X  258GAGAGAAGTCCACCAC 0.98 0.99 0.98 0.99 SeqID 70 X  259 GAGAGAAGTCCACCA0.98 0.99 0.98 0.99 SeqID 71 X  259 TGAGAGAAGTCCACCA 0.98 0.99 0.98 0.99SeqID 72 X  260 GAGAGAAGTCCACC 0.98 0.99 0.98 0.99 SeqID 73 X  260TGAGAGAAGTCCACC 0.98 0.99 0.98 0.99 SeqID 74 X  261 TGAGAGAAGTCCAC 0.990.96 0.95 0.98 SeqID 75 X  384 AAAACGCCGCAGA 0.98 0.96 0.95 0.98SeqID 76 X  384 TAAAACGCCGCAGA 0.98 0.96 0.95 0.97 SeqID 77 X  384ATAAAACGCCGCAGA 0.98 0.96 0.95 0.97 SeqID 78 X  384 GATAAAACGCCGCAGA0.98 0.97 0.96 0.97 SeqID 79 X  385 ATAAAACGCCGCAG 0.98 0.97 0.96 0.97SeqID 80 X  385 GATAAAACGCCGCAG 0.98 0.97 0.96 0.97 SeqID 81 X  385TGATAAAACGCCGCAG 0.98 0.97 0.96 0.98 SeqID 82 X  386 ATAAAACGCCGCA 0.980.97 0.96 0.98 SeqID 83 X  386 GATAAAACGCCGCA 0.98 0.97 0.96 0.98SeqID 84 X  386 TGATAAAACGCCGCA 0.98 0.97 0.96 0.98 SeqID 85 X  386ATGATAAAACGCCGCA 0.98 0.98 0.97 0.98 SeqID 86 X  387 ATAAAACGCCGC 0.980.98 0.97 0.98 SeqID 87 X  387 GATAAAACGCCGC 0.98 0.97 0.97 0.98SeqID 88 X  387 TGATAAAACGCCGC 0.98 0.97 0.97 0.98 SeqID 89 X  387ATGATAAAACGCCGC 0.98 0.98 0.97 0.98 SeqID 90 X  388 GATAAAACGCCG 0.980.97 0.97 0.98 SeqID 91 X  388 TGATAAAACGCCG 0.98 0.97 0.97 0.98SeqID 92 X  388 ATGATAAAACGCCG 0.98 0.97 0.97 0.98 SeqID 93 X  389TGATAAAACGCC 0.98 0.97 0.97 0.98 SeqID 94 X  389 ATGATAAAACGCC 0.98 0.970.97 0.98 SeqID 95 X  390 ATGATAAAACGC 1.00 0.99 0.98 0.97 SeqID 96 X 411 TAGCAGCAGGATG 1.00 0.99 0.98 0.97 SeqID 97 X  411 ATAGCAGCAGGATG1.00 0.99 0.98 0.97 SeqID 98 X  411 CATAGCAGCAGGATG 1.00 0.98 0.98 0.97SeqID 99 X  411 GCATAGCAGCAGGATG 1.00 0.99 0.98 0.97 SeqID 100 X  412GCATAGCAGCAGGAT 1.00 0.99 0.98 0.97 SeqID 101 X  412 GGCATAGCAGCAGGAT0.99 0.98 0.98 0.97 SeqID 102 X  414 GAGGCATAGCAGCAGG 0.99 0.98 0.980.97 SeqID 103 X  415 TGAGGCATAGCAGCAG 0.99 0.98 0.98 0.97 SeqID 104 X 416 TGAGGCATAGCAGCA 0.99 0.97 0.96 0.96 SeqID 105 X  416ATGAGGCATAGCAGCA 0.99 0.98 0.98 0.97 SeqID 106 X  417 TGAGGCATAGCAGC0.99 0.97 0.96 0.96 SeqID 107 X  417 ATGAGGCATAGCAGC 0.99 0.97 0.96 0.96SeqID 108 X  417 GATGAGGCATAGCAGC 1.00 0.97 0.96 0.96 SeqID 109 X  418GATGAGGCATAGCAG 1.00 0.97 0.96 0.96 SeqID 110 X  418 AGATGAGGCATAGCAG1.00 0.97 0.97 0.96 SeqID 111 X  419 GATGAGGCATAGCA 1.00 0.97 0.97 0.96SeqID 112 X  419 AGATGAGGCATAGCA 1.00 0.97 0.97 0.96 SeqID 113 X  419AAGATGAGGCATAGCA 1.00 0.96 0.97 0.98 SeqID 114 X  422 AAGAAGATGAGGCATA1.00 0.96 0.97 0.98 SeqID 115 X  423 AAGAAGATGAGGCAT 0.98 0.99 1.00 0.99SeqID 116 X  601 TGGGATGGGAATACA 0.98 0.99 0.99 0.99 SeqID 117 X  601ATGGGATGGGAATACA 0.98 0.99 1.00 0.99 SeqID 118 X  602 TGGGATGGGAATAC0.98 0.99 1.00 0.99 SeqID 119 X  602 ATGGGATGGGAATAC 0.97 0.99 0.99 0.99SeqID 120 X  602 GATGGGATGGGAATAC 0.98 0.99 1.00 0.99 SeqID 121 X  603ATGGGATGGGAATA 0.97 0.99 0.99 0.99 SeqID 122 X  603 GATGGGATGGGAATA 0.970.99 0.99 0.99 SeqID 123 X  604 GATGGGATGGGAAT 0.99 0.97 0.96 0.98SeqID 834  670 TAGTAAACTGAGCCA SeqID 835  670 CTAGTAAACTGAGCCA SeqID 836 671 CTAGTAAACTGAGCC SeqID 837  674 GCACTAGTAAACTGA SeqID 838  674GGCACTAGTAAACTGA SeqID 124 X  691 AACCACTGAACAAA 0.99 0.97 0.96 0.98SeqID 4 X  691 GAACCACTGAACAAA 0.99 0.97 0.96 0.98 SeqID 5 X  691CGAACCACTGAACAAA 0.99 0.97 0.96 0.98 SeqID 6 X  692 CGAACCACTGAACAA 0.990.97 0.96 0.98 SeqID 7 X  693 CGAACCACTGAACA 0.99 0.98 0.96 0.98 SeqID 8X  694 CGAACCACTGAAC 0.99 0.98 0.96 0.98 SeqID 125 X  695 CGAACCACTGAA0.98 0.97 0.98 0.98 SeqID 126 X  708 GGGGGAAAGCCCT 0.97 0.97 0.98 0.98SeqID 127 X  708 TGGGGGAAAGCCCT 0.99 0.97 0.97 0.99 SeqID 839 1141CAACGGGGTAAAGGT SeqID 128 X 1142 GCAACGGGGTAAAGG 0.99 0.97 0.97 0.99SeqID 129 X 1143 GCAACGGGGTAAAG 0.99 0.97 0.97 0.99 SeqID 130 X 1144GCAACGGGGTAAA 0.99 0.98 0.97 0.99 SeqID 131 X 1176 AGCAAACACTTGGCA 0.990.98 0.97 0.99 SeqID 132 X 1176 CAGCAAACACTTGGCA 0.99 0.98 0.97 0.99SeqID 133 X 1177 CAGCAAACACTTGGC 0.99 0.98 0.97 0.99 SeqID 134 X 1177TCAGCAAACACTTGGC 0.99 0.98 0.97 0.99 SeqID 135 X 1178 TCAGCAAACACTTGG0.98 0.96 0.96 0.97 SeqID 840 1261 CAGTATGGATCGGCA SeqID 136 x 1264GCAGTATGGATCG 0.98 0.95 0.96 0.97 SeqID 137 x 1264 CGCAGTATGGATCG 0.980.95 0.96 0.97 SeqID 9 x 1264 CCGCAGTATGGATCG 0.98 0.95 0.96 0.97SeqID 138 x 1264 TCCGCAGTATGGATCG 0.98 0.95 0.96 0.97 SeqID 832 1264CCGCAGTATGGATCG SeqID 10 x 1265 CGCAGTATGGATC 0.98 0.95 0.96 0.97SeqID 139 x 1265 CCGCAGTATGGATC 0.98 0.95 0.96 0.97 SeqID 140 x 1265TCCGCAGTATGGATC 0.99 0.96 0.97 0.97 SeqID 841 1265 TTCCGCAGTATGGATCSeqID 842 1266 TTCCGCAGTATGGAT SeqID 843 1266 GTTCCGCAGTATGGAT SeqID 141x 1266 CGCAGTATGGAT 0.99 0.96 0.97 0.97 SeqID 142 x 1266 CCGCAGTATGGAT0.99 0.96 0.97 0.97 SeqID 143 x 1266 TCCGCAGTATGGAT 0.99 0.96 0.97 0.97SeqID 144 x 1267 TCCGCAGTATGGA 0.99 0.95 0.97 0.97 SeqID 844 1267GTTCCGCAGTATGGA SeqID 845 1267 AGTTCCGCAGTATGGA SeqID 846 1268AGTTCCGCAGTATGG SeqID 847 1268 GAGTTCCGCAGTATGG SeqID 848 1269GAGTTCCGCAGTATG SeqID 849 1269 GGAGTTCCGCAGTATG SeqID 145 x 1269TTCCGCAGTATG 0.99 0.99 0.99 0.99 SeqID 850 1525 TAAAGAGAGGTGCGCCSeqID 851 1526 TAAAGAGAGGTGCGC SeqID 852 1526 GTAAAGAGAGGTGCGC SeqID 8531527 GTAAAGAGAGGTGCG SeqID 854 1527 CGTAAAGAGAGGTGCG SeqID 855 1528CGTAAAGAGAGGTGC SeqID 856 1528 GCGTAAAGAGAGGTGC SeqID 857 1529GCGTAAAGAGAGGTG SeqID 858 1529 CGCGTAAAGAGAGGTG SeqID 146 x 1530CGTAAAGAGAGGT 0.99 0.98 0.99 0.99 SeqID 11 x 1530 GCGTAAAGAGAGGT 0.990.98 0.99 0.99 SeqID 12 x 1530 CGCGTAAAGAGAGGT 0.99 0.98 0.99 0.99SeqID 147 x 1530 CCGCGTAAAGAGAGGT 0.99 0.98 0.99 0.99 SeqID 148 x 1531CGTAAAGAGAGG 0.99 0.98 0.99 0.99 SeqID 13 x 1531 GCGTAAAGAGAGG 0.99 0.980.99 0.99 SeqID 149 x 1531 CGCGTAAAGAGAGG 0.99 0.98 0.99 0.99 SeqID 150x 1531 CCGCGTAAAGAGAGG 0.99 0.98 0.99 1.00 SeqID 151 x 1532CGCGTAAAGAGAG 0.99 0.98 0.99 1.00 SeqID 152 x 1532 CCGCGTAAAGAGAG 0.990.98 0.99 1.00 SeqID 153 x 1533 CGCGTAAAGAGA 0.99 0.98 0.99 1.00SeqID 154 x 1533 CCGCGTAAAGAGA 0.99 0.99 0.99 1.00 SeqID 155 x 1534CCGCGTAAAGAG 0.98 0.98 0.99 1.00 SeqID 156 x 1547 GGCACAGACGGGGAG 0.980.98 0.99 1.00 SeqID 157 x 1547 AGGCACAGACGGGGAG 0.98 0.99 0.99 1.00SeqID 158 x 1548 GGCACAGACGGGGA 0.98 0.98 0.99 1.00 SeqID 159 x 1548AGGCACAGACGGGGA 0.98 0.98 0.99 1.00 SeqID 160 x 1548 AAGGCACAGACGGGGA0.98 0.98 0.99 1.00 SeqID 161 x 1549 AGGCACAGACGGGG 0.98 0.98 0.99 1.00SeqID 162 x 1549 AAGGCACAGACGGGG 0.98 0.97 0.99 0.99 SeqID 163 x 1549GAAGGCACAGACGGGG 0.97 0.98 0.98 0.98 SeqID 164 x 1550 AGAAGGCACAGACGGG0.97 0.98 0.98 0.98 SeqID 14 x 1551 AGAAGGCACAGACGG 0.97 0.98 0.98 0.98SeqID 15 x 1551 GAGAAGGCACAGACGG 0.97 0.98 0.98 0.98 SeqID 165 x 1552GAGAAGGCACAGACG 0.99 0.99 0.99 0.98 SeqID 859 1552 TGAGAAGGCACAGACGSeqID 16 x 1577 GAAGTGCACACGG 0.99 0.99 0.99 0.98 SeqID 166 x 1577CGAAGTGCACACGG 0.98 0.99 0.99 0.98 SeqID 17 x 1577 GCGAAGTGCACACGG 0.980.99 0.99 0.96 SeqID 18 x 1577 AGCGAAGTGCACACGG 0.99 0.99 0.99 0.98SeqID 19 x 1578 CGAAGTGCACACG 0.98 0.99 0.99 0.98 SeqID 167 x 1578GCGAAGTGCACACG 0.98 0.99 0.99 0.96 SeqID 20 x 1578 AGCGAAGTGCACACG 0.980.99 0.99 0.96 SeqID 21 x 1578 AAGCGAAGTGCACACG 0.98 0.99 0.99 0.98SeqID 168 x 1579 GCGAAGTGCACAC 0.98 0.99 0.99 0.96 SeqID 169 x 1579AGCGAAGTGCACAC 0.98 0.99 0.99 0.96 SeqID 170 x 1579 AAGCGAAGTGCACAC 0.980.99 0.99 0.96 SeqID 171 x 1579 GAAGCGAAGTGCACAC 0.98 0.99 0.99 0.96SeqID 172 x 1580 AGCGAAGTGCACA 0.98 0.99 0.99 0.96 SeqID 173 x 1580AAGCGAAGTGCACA 0.98 0.99 0.99 0.96 SeqID 22 x 1580 GAAGCGAAGTGCACA 0.980.99 0.99 0.96 SeqID 174 x 1580 TGAAGCGAAGTGCACA 0.98 0.99 0.99 0.96SeqID 175 x 1581 AAGCGAAGTGCAC 0.98 0.99 0.99 0.96 SeqID 176 x 1581GAAGCGAAGTGCAC 0.98 0.99 0.99 0.96 SeqID 177 x 1581 TGAAGCGAAGTGCAC 0.980.99 0.99 0.96 SeqID 178 x 1581 GTGAAGCGAAGTGCAC 0.98 0.99 0.99 0.96SeqID 179 x 1582 AAGCGAAGTGCA 0.98 0.99 0.99 0.96 SeqID 180 x 1582GAAGCGAAGTGCA 0.98 0.99 0.99 0.96 SeqID 181 x 1582 TGAAGCGAAGTGCA 0.980.99 0.99 0.96 SeqID 182 x 1582 GTGAAGCGAAGTGCA 0.98 0.99 0.99 0.96SeqID 23 x 1582 GGTGAAGCGAAGTGCA 0.98 0.99 0.99 0.96 SeqID 183 x 1583TGAAGCGAAGTGC 0.98 0.99 0.99 0.96 SeqID 184 x 1583 GTGAAGCGAAGTGC 0.980.99 0.99 0.96 SeqID 24 x 1583 GGTGAAGCGAAGTGC 0.98 0.99 0.99 0.96SeqID 25 x 1583 AGGTGAAGCGAAGTGC 0.98 0.99 0.99 0.96 SeqID 185 x 1584GTGAAGCGAAGTG 0.98 0.99 0.99 0.96 SeqID 186 x 1584 GGTGAAGCGAAGTG 0.980.99 0.99 0.96 SeqID 26 x 1584 AGGTGAAGCGAAGTG 0.98 0.99 0.99 0.96SeqID 187 x 1584 GAGGTGAAGCGAAGTG 0.98 0.99 0.99 0.96 SeqID 188 x 1585GTGAAGCGAAGT 0.98 0.99 0.99 0.96 SeqID 189 x 1585 GGTGAAGCGAAGT 0.980.99 0.99 0.96 SeqID 27 x 1585 AGGTGAAGCGAAGT 0.98 0.99 0.99 0.96SeqID 190 x 1585 GAGGTGAAGCGAAGT 0.98 0.99 0.99 0.96 SeqID 191 x 1585AGAGGTGAAGCGAAGT 0.98 0.99 0.99 0.96 SeqID 192 x 1586 AGAGGTGAAGCGAAG0.98 0.99 0.99 0.96 SeqID 193 x 1586 CAGAGGTGAAGCGAAG 0.99 0.99 0.990.96 SeqID 194 x 1587 AGAGGTGAAGCGAA 0.99 0.99 0.99 0.96 SeqID 195 x1587 CAGAGGTGAAGCGAA 0.99 0.99 0.98 0.96 SeqID 196 x 1587GCAGAGGTGAAGCGAA 0.99 0.99 0.99 0.97 SeqID 28 x 1588 CAGAGGTGAAGCGA 0.990.99 0.98 0.96 SeqID 197 x 1588 GCAGAGGTGAAGCGA 0.99 0.99 0.98 0.96SeqID 198 x 1588 TGCAGAGGTGAAGCGA 0.99 0.99 0.98 0.96 SeqID 199 x 1589TGCAGAGGTGAAGCG 0.99 0.99 0.98 0.96 SeqID 200 x 1589 GTGCAGAGGTGAAGCG0.99 0.99 0.98 0.96 SeqID 201 x 1590 CGTGCAGAGGTGAAGC 0.99 0.99 0.980.96 SeqID 202 x 1591 CGTGCAGAGGTGAAG 0.99 0.99 0.98 0.96 SeqID 203 x1591 ACGTGCAGAGGTGAAG 1.00 0.99 0.98 0.96 SeqID 204 x 1592CGTGCAGAGGTGAA 1.00 0.99 0.98 0.96 SeqID 205 x 1592 ACGTGCAGAGGTGAA 1.000.99 0.98 0.99 SeqID 206 x 1593 CGTGCAGAGGTGA 1.00 0.99 0.98 0.99SeqID 207 x 1593 ACGTGCAGAGGTGA 0.98 0.97 0.96 0.97 SeqID 208 x 1616CGTTCACGGTGGT 0.98 0.96 0.96 0.95 SeqID 209 x 1690 CTCAAGGTCGGTC 0.990.97 0.98 0.96 SeqID 860 1690 GCCTCAAGGTCGGTC SeqID 210 x 1691CCTCAAGGTCGGT 0.99 0.97 0.98 0.95 SeqID 211 x 1691 GCCTCAAGGTCGGT 0.980.97 0.98 0.99 SeqID 212 x 1706 ACAGTCTTTGAAGTA 0.99 0.95 0.95 0.99SeqID 861 1778 ATGCCTACAGCCTCC SeqID 213 x 1783 TTTATGCCTACAG 0.99 0.960.95 0.99 SeqID 214 x 1784 AATTTATGCCTACA 0.99 0.96 0.95 0.99 SeqID 215x 1785 AATTTATGCCTAC 0.99 0.95 0.96 0.99 SeqID 862 1785 ACCAATTTATGCCTACSeqID 216 x 1787 CCAATTTATGCCT 0.97 0.99 0.99 0.98 SeqID 217 x 1865GCTTGGAGGCTTGAA 0.97 0.99 0.98 0.98 SeqID 218 x 1865 AGCTTGGAGGCTTGAA0.97 0.99 0.99 0.98 SeqID 219 x 1866 GCTTGGAGGCTTGA 0.97 0.99 0.99 0.98SeqID 220 x 1866 AGCTTGGAGGCTTGA 0.97 0.98 0.98 0.98 SeqID 221 x 1866CAGCTTGGAGGCTTGA 0.97 0.99 0.99 0.98 SeqID 222 x 1867 GCTTGGAGGCTTG 0.970.99 0.99 0.98 SeqID 223 x 1867 AGCTTGGAGGCTTG 0.97 0.98 0.98 0.98SeqID 224 x 1867 CAGCTTGGAGGCTTG 0.97 0.98 0.98 0.98 SeqID 225 x 1867ACAGCTTGGAGGCTTG 0.97 0.98 0.98 0.98 SeqID 226 x 1868 CACAGCTTGGAGGCTT0.97 0.98 0.98 0.98 SeqID 227 x 1869 CACAGCTTGGAGGCT 0.97 0.98 0.98 0.98SeqID 228 x 1869 GCACAGCTTGGAGGCT 0.97 0.98 0.98 0.98 SeqID 229 x 1870GCACAGCTTGGAGGC 0.97 0.98 0.98 0.98 SeqID 230 x 1870 GGCACAGCTTGGAGGC0.96 0.98 0.98 0.98 SeqID 231 x 1871 AGGCACAGCTTGGAGG 0.96 0.98 0.980.99 SeqID 232 x 1872 AGGCACAGCTTGGAG 0.96 0.97 0.98 0.99 SeqID 233 x1872 AAGGCACAGCTTGGAG 0.96 0.97 0.98 0.99 SeqID 234 x 1873AAGGCACAGCTTGGA 0.96 0.97 0.98 0.98 SeqID 235 x 1873 CAAGGCACAGCTTGGA0.96 0.97 0.98 0.99 SeqID 236 x 1874 AAGGCACAGCTTGG 0.96 0.97 0.98 0.99SeqID 237 x 1874 CAAGGCACAGCTTGG 0.96 0.97 0.97 0.98 SeqID 238 x 1874CCAAGGCACAGCTTGG 0.96 0.97 0.98 0.99 SeqID 239 x 1875 CAAGGCACAGCTTG0.96 0.97 0.97 0.98 SeqID 240 x 1875 CCAAGGCACAGCTTG 0.96 0.97 0.97 0.99SeqID 241 x 1876 CCAAGGCACAGCTT 0.96 0.97 0.96 0.97 SeqID 242 2272TGCGAATCCACAC 0.96 0.97 0.96 0.97 SeqID 243 2272 GTGCGAATCCACAC 0.960.96 0.98 0.97 SeqID 244 2370 GGAGTTCTTCTTCTA 0.96 0.96 0.98 0.97SeqID 245 2370 GGGAGTTCTTCTTCTA 0.96 0.96 0.98 0.97 SeqID 246 2371GGGAGTTCTTCTTCT 0.96 0.96 0.98 0.97 SeqID 247 2371 AGGGAGTTCTTCTTCT 0.960.98 0.98 0.97 SeqID 248 2372 AGGGAGTTCTTCTTC 0.96 0.98 0.97 0.97SeqID 249 2372 GAGGGAGTTCTTCTTC 0.97 0.98 0.98 0.97 SeqID 250 2373AGGGAGTTCTTCTT 0.97 0.98 0.98 0.97 SeqID 251 2373 GAGGGAGTTCTTCTT 0.970.95 0.97 0.95 SeqID 252 2373 CGAGGGAGTTCTTCTT 0.97 0.95 0.97 0.96SeqID 253 2374 CGAGGGAGTTCTTCT 0.97 0.95 0.97 0.96 SeqID 254 2374GCGAGGGAGTTCTTCT 0.97 0.96 0.97 0.96 SeqID 255 2375 GCGAGGGAGTTCTTC 0.970.96 0.97 0.96 SeqID 256 2375 GGCGAGGGAGTTCTTC 0.97 0.96 0.97 0.96SeqID 257 2376 GCGAGGGAGTTCTT 0.97 0.96 0.97 0.96 SeqID 258 2376GGCGAGGGAGTTCTT 0.97 0.96 0.97 0.96 SeqID 259 2376 AGGCGAGGGAGTTCTT 0.980.96 0.97 0.96 SeqID 260 2377 GCGAGGGAGTTCT 0.98 0.96 0.97 0.96SeqID 261 2377 GGCGAGGGAGTTCT 0.98 0.96 0.97 0.96 SeqID 262 2377AGGCGAGGGAGTTCT 0.98 0.96 0.97 0.96 SeqID 263 2377 GAGGCGAGGGAGTTCT 0.990.96 0.98 0.97 SeqID 264 2378 GGCGAGGGAGTTC 0.99 0.96 0.98 0.97SeqID 265 2378 AGGCGAGGGAGTTC 0.99 0.96 0.98 0.97 SeqID 266 2378GAGGCGAGGGAGTTC 0.99 0.96 0.97 0.97 SeqID 267 2378 CGAGGCGAGGGAGTTC 0.990.96 0.98 0.97 SeqID 268 2379 AGGCGAGGGAGTT 0.99 0.96 0.98 0.97SeqID 269 2379 GAGGCGAGGGAGTT 0.99 0.96 0.98 0.97 SeqID 270 2379CGAGGCGAGGGAGTT 0.99 0.96 0.98 0.97 SeqID 271 2379 GCGAGGCGAGGGAGTT 0.990.96 0.98 0.97 SeqID 272 2380 GAGGCGAGGGAGT 0.99 0.96 0.98 0.97SeqID 273 2380 CGAGGCGAGGGAGT 0.99 0.96 0.98 0.97 SeqID 274 2380GCGAGGCGAGGGAGT 0.99 0.96 0.97 0.96 SeqID 275 2380 TGCGAGGCGAGGGAGT 0.990.96 0.98 0.97 SeqID 276 2381 CGAGGCGAGGGAG 0.99 0.96 0.98 0.97SeqID 277 2381 GCGAGGCGAGGGAG 0.99 0.96 0.97 0.97 SeqID 278 2381TGCGAGGCGAGGGAG 0.97 0.96 0.96 0.96 SeqID 279 2381 CTGCGAGGCGAGGGAG 0.990.96 0.98 0.97 SeqID 280 2382 CGAGGCGAGGGA 0.99 0.96 0.98 0.97 SeqID 2812382 GCGAGGCGAGGGA 0.99 0.96 0.97 0.97 SeqID 282 2382 TGCGAGGCGAGGGA0.97 0.96 0.96 0.96 SeqID 283 2382 CTGCGAGGCGAGGGA 0.97 0.96 0.96 0.96SeqID 284 2382 TCTGCGAGGCGAGGGA 0.97 0.96 0.96 0.96 SeqID 285 2383TCTGCGAGGCGAGGG 0.97 0.96 0.96 0.96 SeqID 286 2383 GTCTGCGAGGCGAGGG 0.980.97 0.96 0.95 SeqID 287 2824 GTTCCCAAGAATAT 0.98 0.97 0.96 0.95SeqID 288 2824 TGTTCCCAAGAATAT 0.98 0.98 0.97 0.96 SeqID 289 2825GTTCCCAAGAATA 0.98 0.98 0.97 0.96 SeqID 290 2825 TGTTCCCAAGAATA 0.970.97 0.96 0.96 SeqID 291 2825 TTGTTCCCAAGAATA 0.98 0.98 0.97 0.96SeqID 292 2826 TGTTCCCAAGAAT 0.97 0.97 0.96 0.96 SeqID 293 2826TTGTTCCCAAGAAT

TABLE 2 A subset of oligomer motifs from table 1 Start position SEQTarget Target on ID No. HBx HBsAg U95551 Oligo_seq SeqID 4 X  691GAACCACTGAACAAA SeqID 5 X  691 CGAACCACTGAACAAA SeqID 6 X  692CGAACCACTGAACAA SeqID 7 X  693 CGAACCACTGAACA SeqID 8 X  694CGAACCACTGAAC SeqID 9 X 1264 CCGCAGTATGGATCG SeqID 10 X 1265CGCAGTATGGATC SeqID 11 X 1530 GCGTAAAGAGAGGT SeqID 12 X 1530CGCGTAAAGAGAGGT SeqID 13 X 1531 GCGTAAAGAGAGG SeqID 14 X 1551AGAAGGCACAGACGG SeqID 15 X 1551 GAGAAGGCACAGACGG SeqID 16 X 1577GAAGTGCACACGG SeqID 17 X 1577 GCGAAGTGCACACGG SeqID 18 X 1577AGCGAAGTGCACACGG SeqID 19 X 1578 CGAAGTGCACACG SeqID 20 X 1578AGCGAAGTGCACACG SeqID 21 X 1578 AAGCGAAGTGCACACG SeqID 22 X 1580GAAGCGAAGTGCACA SeqID 23 X 1582 GGTGAAGCGAAGTGCA SeqID 24 X 1583GGTGAAGCGAAGTGC SeqID 25 X 1583 AGGTGAAGCGAAGTGC SeqID 26 X 1584AGGTGAAGCGAAGTG SeqID 27 X 1585 AGGTGAAGCGAAGT SeqID 28 X 1588CAGAGGTGAAGCGA

TABLE 3 LNA oligomers Upper case letters denote beta-D-oxy LNA,C LNA is 5-methyl C LNA, lower case lettersdenote DNA, ^(m)c denotes a 5-methylcytosineDNA, all internucleoside linkages arephosphorothiate internucleoside linkages. Start position SEQ TargetTarget on ID No. HBx HBsAg U95551 Oligo_seq SeqID 294 x  691GAAccactgaacAAA SeqID 295 x  691 CGAaccactgaacAAA SeqID 296 x  692CGAaccactgaaCAA SeqID 297 x  693 CGAaccactgaACA SeqID 298 x  694CGAaccactgaAC SeqID 299 x 1264 CCGcagtatggaTCG SeqID 300 x 1265CGCagtatggaTC SeqID 301 x 1530 GCGtaaagagaGGT SeqID 302 x 1530CGCgtaaagagaGGT SeqID 303 x 1531 GCGtaaagagaGG SeqID 304 x 1551AGAaggcacagaCGG SeqID 305 x 1551 GAGaaggcacagaCGG SeqID 306 x 1577GAAgtgcaca^(m)cGG SeqID 307 x 1577 GCGaagtgcacaCGG SeqID 308 x 1577AGCgaagtgcacaCGG SeqID 309 x 1578 CGAagtgcacaCG SeqID 310 x 1578AGCgaagtgcacACG SeqID 311 x 1578 AAG^(m)cgaagtgcacACG SeqID 312 x 1580GAAg^(m)cgaagtgcACA SeqID 313 x 1582 GGTgaag^(m)cgaagtGCA SeqID 314 x1583 GGTgaag^(m)cgaagTGC SeqID 315 x 1583 AGGtgaag^(m)cgaagTGC SeqID 316x 1584 AGGtgaag^(m)cgaaGTG SeqID 317 x 1585 AGGtgaag^(m)cgaAGT SeqID 318x 1588 CAGaggtgaagCGA SeqID 319 x  201 AAAaccc^(m)cgccTGT SeqID 320 x 202 AAAaccc^(m)cgccTG SeqID 321 x  245 ACGagtctagacTCT SeqID 322 x  245CACgagtctagacTCT SeqID 323 x  246 ACGagtctagaCTC SeqID 324 x  246CACgagtctagaCTC SeqID 325 x  246 CCA^(m)cgagtctagaCTC SeqID 326 x  247ACGagtctagaCT SeqID 327 x  247 CACgagtctagACT SeqID 328 x  247CCA^(m)cgagtctagACT SeqID 329 x  247 ACCa^(m)cgagtctagACT SeqID 330 x 248 ACgagtctagAC SeqID 331 x  248 CACgagtctagAC SeqID 332 x  248CCA^(m)cgagtctaGAC SeqID 333 x  248 ACCa^(m)cgagtctaGAC SeqID 334 x  248CACca^(m)cgagtctaGAC SeqID 335 x  249 ACCa^(m)cgagtctAGA SeqID 336 x 249 CACca^(m)cgagtctAGA SeqID 337 x  249 CCAcca^(m)cgagtctAGA SeqID 338x  250 CCAcca^(m)cgagtcTAG SeqID 339 x  250 TCCacca^(m)cgagtcTAGSeqID 340 x  251 CCAcca^(m)cgagtCTA SeqID 341 x  251 TCCacca^(m)cgagtCTASeqID 342 x  251 GTCcacca^(m)cgagtCTA SeqID 343 x  252TCCacca^(m)cgagTCT SeqID 344 x  252 GTCcacca^(m)cgagTCT SeqID 345 x  252AGTccacca^(m)cgagTCT SeqID 346 x  253 GTCcacca^(m)cgaGTC SeqID 347 x 253 AGTccacca^(m)cgaGTC SeqID 348 x  253 AAGtccacca^(m)cgaGTC SeqID 349x  254 AGTccacca^(m)cgAGT SeqID 350 x  254 AAGtccacca^(m)cgAGT SeqID 351x  254 GAAgtccacca^(m)cgAGT SeqID 352 x  255 AAGtccacca^(m)cgAGSeqID 353 x  255 GAAgtccacca^(m)cgAG SeqID 354 x  255AGAagtccacca^(m)cgAG SeqID 355 x  256 AGAagtccaccaCGA SeqID 356 x  256GAGaagtccaccaCGA SeqID 357 x  257 GAGaagtccaccACG SeqID 358 x  257AGAgaagtccaccACG SeqID 359 x  258 GAGagaagtccacCAC SeqID 360 x  259GAGagaagtccaCCA SeqID 361 x  259 TGAgagaagtccaCCA SeqID 362 x  260GAGagaagtccACC SeqID 363 x  260 TGAgagaagtccACC SeqID 364 x  261TGAgagaagtcCAC SeqID 365 x  384 AAAa^(m)cgc^(m)cgcaGA SeqID 366 x  384TAAaa^(m)cgc^(m)cgcAGA SeqID 367 x  384 ATAaaa^(m)cgc^(m)cgcAGASeqID 368 x  384 GATaaaa^(m)cgc^(m)cgcAGA SeqID 369 x  385ATAaaa^(m)cgc^(m)cgCAG SeqID 370 x  385 GATaaaa^(m)cgc^(m)cgCAGSeqID 371 x  385 TGAtaaaa^(m)cgc^(m)cgCAG SeqID 372 x  386ATAaaa^(m)cgc^(m)cgCA SeqID 373 x  386 GATaaaa^(m)cgc^(m)cGCA SeqID 374x  386 TGAtaaaa^(m)cgc^(m)cGCA SeqID 375 x  386 ATGataaaa^(m)cgc^(m)cGCASeqID 376 x  387 ATaaaa^(m)cgc^(m)cGC SeqID 377 x  387GATaaaa^(m)cgc^(m)cGC SeqID 378 x  387 TGAtaaaa^(m)cgcCGC SeqID 379 x 387 ATGataaaa^(m)cgcCGC SeqID 380 x  388 GAtaaaa^(m)cgcCG SeqID 381 x 388 TGAtaaaa^(m)cgcCG SeqID 382 x  388 ATGataaaa^(m)cgCCG SeqID 383 x 389 TGataaaa^(m)cgCC SeqID 384 x  389 ATGataaaa^(m)cgCC SeqID 385 x 390 ATgataaaa^(m)cGC SeqID 386 x  411 TAGcagcaggaTG SeqID 387 x  411ATAgcagcaggATG SeqID 388 x  411 CATagcagcaggATG SeqID 389 x  411GCAtagcagcaggATG SeqID 390 x  412 GCAtagcagcagGAT SeqID 391 x  412GGCatagcagcagGAT SeqID 392 x  414 GAGgcatagcagcAGG SeqID 393 x  415TGAggcatagcagCAG SeqID 394 x  416 TGAggcatagcaGCA SeqID 395 x  416ATGaggcatagcaGCA SeqID 396 x  417 TGAggcatagcAGC SeqID 397 x  417ATGaggcatagcAGC SeqID 398 x  417 GATgaggcatagcAGC SeqID 399 x  418GATgaggcatagCAG SeqID 400 x  418 AGAtgaggcatagCAG SeqID 401 x  419GATgaggcataGCA SeqID 402 x  419 AGAtgaggcataGCA SeqID 403 x  419AAGatgaggcataGCA SeqID 404 x  422 AAGaagatgaggcATA SeqID 405 x  423AAGaagatgaggCAT SeqID 406 x  601 TGGgatgggaatACA SeqID 407 x  601ATGggatgggaatACA SeqID 408 x  602 TGGgatgggaaTAC SeqID 409 x  602ATGggatgggaaTAC SeqID 410 x  602 GATgggatgggaaTAC SeqID 411 x  603ATGggatgggaATA SeqID 412 x  603 GATgggatgggaATA SeqID 413 x  604GATgggatgggAAT SeqID 414 x  691 AACcactgaacAAA SeqID 415 x  695CGaaccactgAA SeqID 416 x  708 GGGggaaagccCT SeqID 417 x  708TGGgggaaagcCCT SeqID 418 x 1142 GCAa^(m)cggggtaaAGG SeqID 419 x 1143GCAa^(m)cggggtaAAG SeqID 420 x 1144 GCAa^(m)cggggtaAA SeqID 421 x 1176AGCaaacacttgGCA SeqID 422 x 1176 CAGcaaacacttgGCA SeqID 423 x 1177CAGcaaacacttGGC SeqID 424 x 1177 TCAgcaaacacttGGC SeqID 425 x 1178TCAgcaaacactTGG SeqID 426 x 1264 GCAgtatggatCG SeqID 427 x 1264CGCagtatggaTCG SeqID 428 x 1264 TCCgcagtatggaTCG SeqID 429 x 1265CCGcagtatggATC SeqID 430 x 1265 TCCgcagtatggATC SeqID 431 x 1266CGcagtatggAT SeqID 432 x 1266 CCGcagtatggAT SeqID 433 x 1266TCCgcagtatgGAT SeqID 434 x 1267 TCCgcagtatgGA SeqID 435 x 1269TTc^(m)cgcagtaTG SeqID 436 x 1530 CGTaaagagagGT SeqID 437 x 1530CCG^(m)cgtaaagagaGGT SeqID 438 x 1531 CGtaaagagaGG SeqID 439 x 1531CGCgtaaagagAGG SeqID 440 x 1531 CCG^(m)cgtaaagagAGG SeqID 441 x 1532CGCgtaaagagAG SeqID 442 x 1532 CCG^(m)cgtaaagaGAG SeqID 443 x 1533CG^(m)cgtaaagaGA SeqID 444 x 1533 CCG^(m)cgtaaagaGA SeqID 445 x 1534CCg^(m)cgtaaagAG SeqID 446 x 1547 GGCacaga^(m)cgggGAG SeqID 447 x 1547AGGcacaga^(m)cgggGAG SeqID 448 x 1548 GGCacaga^(m)cggGGA SeqID 449 x1548 AGGcacaga^(m)cggGGA SeqID 450 x 1548 AAGgcacaga^(m)cggGGA SeqID 451x 1549 AGGcacaga^(m)cgGGG SeqID 452 x 1549 AAGgcacaga^(m)cgGGG SeqID 453x 1549 GAAggcacaga^(m)cgGGG SeqID 454 x 1550 AGAaggcacaga^(m)cGGGSeqID 455 x 1552 GAGaaggcacagACG SeqID 456 x 1577 CGAagtgcacaCGGSeqID 457 x 1578 GCGaagtgcacACG SeqID 458 x 1579 GCGaagtgcacAC SeqID 459x 1579 AGCgaagtgcaCAC SeqID 460 x 1579 AAG^(m)cgaagtgcaCAC SeqID 461 x1579 GAAg^(m)cgaagtgcaCAC SeqID 462 x 1580 AGCgaagtgcaCA SeqID 463 x1580 AAG^(m)cgaagtgcACA SeqID 464 x 1580 TGAag^(m)cgaagtgcACA SeqID 465x 1581 AAG^(m)cgaagtgcAC SeqID 466 x 1581 GAAg^(m)cgaagtgCAC SeqID 467 x1581 TGAag^(m)cgaagtgCAC SeqID 468 x 1581 GTGaag^(m)cgaagtgCAC SeqID 469x 1582 AAg^(m)cgaagtgCA SeqID 470 x 1582 GAAg^(m)cgaagtgCA SeqID 471 x1582 TGAag^(m)cgaagtGCA SeqID 472 x 1582 GTGaag^(m)cgaagtGCA SeqID 473 x1583 TGAag^(m)cgaagtGC SeqID 474 x 1583 GTGaag^(m)cgaagTGC SeqID 475 x1584 GTGaag^(m)cgaagTG SeqID 476 x 1584 GGTgaag^(m)cgaaGTG SeqID 477 x1584 GAGgtgaag^(m)cgaaGTG SeqID 478 x 1585 GTgaag^(m)cgaaGT SeqID 479 x1585 GGTgaag^(m)cgaaGT SeqID 480 x 1585 GAGgtgaag^(m)cgaAGT SeqID 481 x1585 AGAggtgaag^(m)cgaAGT SeqID 482 x 1586 AGAggtgaag^(m)cgAAG SeqID 483x 1586 CAGaggtgaag^(m)cgAAG SeqID 484 x 1587 AGAggtgaag^(m)cGAASeqID 485 x 1587 CAGaggtgaag^(m)cGAA SeqID 486 x 1587GCAgaggtgaag^(m)cGAA SeqID 487 x 1588 GCAgaggtgaagCGA SeqID 488 x 1588TGCagaggtgaagCGA SeqID 489 x 1589 TGCagaggtgaaGCG SeqID 490 x 1589GTGcagaggtgaaGCG SeqID 491 x 1590 CGTgcagaggtgaAGC SeqID 492 x 1591CGTgcagaggtgAAG SeqID 493 x 1591 ACGtgcagaggtgAAG SeqID 494 x 1592CGTgcagaggtGAA SeqID 495 x 1592 ACGtgcagaggtGAA SeqID 496 X 1593CGTgcagaggtGA SeqID 497 x 1593 ACGtgcagaggTGA SeqID 498 x 1616CGTtca^(m)cggtgGT SeqID 499 x 1690 CTCaaggt^(m)cggTC SeqID 500 x 1691CCTcaaggt^(m)cgGT SeqID 501 x 1691 GCCtcaaggt^(m)cGGT SeqID 502 x 1706ACAgtctttgaaGTA SeqID 503 x 1783 TTTatgcctacAG SeqID 504 x 1784AATttatgcctACA SeqID 505 x 1785 AATttatgcctAC SeqID 506 x 1787CCAatttatgcCT SeqID 507 x 1865 GCTtggaggcttGAA SeqID 508 x 1865AGCttggaggcttGAA SeqID 509 x 1866 GCTtggaggctTGA SeqID 510 x 1866AGCttggaggctTGA SeqID 511 x 1866 CAGcttggaggctTGA SeqID 512 x 1867GCTtggaggctTG SeqID 513 x 1867 AGCttggaggcTTG SeqID 514 x 1867CAGcttggaggcTTG SeqID 515 x 1867 ACAgcttggaggcTTG SeqID 516 x 1868CACagcttggaggCTT SeqID 517 x 1869 CACagcttggagGCT SeqID 518 x 1869GCAcagcttggagGCT SeqID 519 x 1870 GCAcagcttggaGGC SeqID 520 x 1870GGCacagcttggaGGC SeqID 521 x 1871 AGGcacagcttggAGG SeqID 522 x 1872AGGcacagcttgGAG SeqID 523 x 1872 AAGgcacagcttgGAG SeqID 524 x 1873AAGgcacagcttGGA SeqID 525 x 1873 CAAggcacagcttGGA SeqID 526 x 1874AAGgcacagctTGG SeqID 527 x 1874 CAAggcacagctTGG SeqID 528 x 1874CCAaggcacagctTGG SeqID 529 x 1875 CAAggcacagcTTG SeqID 530 x 1875CCAaggcacagcTTG SeqID 531 x 1876 CCAaggcacagCTT SeqID 532 2272TGCgaatccacAC SeqID 533 2272 GTG^(m)cgaatccaCAC SeqID 534 2370GGAgttcttcttCTA SeqID 535 2370 GGGagttcttcttCTA SeqID 536 2371GGGagttcttctTCT SeqID 537 2371 AGGgagttcttctTCT SeqID 538 2372AGGgagttcttcTTC SeqID 539 2372 GAGggagttcttcTTC SeqID 540 2373AGGgagttcttCTT SeqID 541 2373 GAGggagttcttCTT SeqID 542 2373CGAgggagttcttCTT SeqID 543 2374 CGAgggagttctTCT SeqID 544 2374GCGagggagttctTCT SeqID 545 2375 GCGagggagttcTTC SeqID 546 2375GGCgagggagttcTTC SeqID 547 2376 GCGagggagttCTT SeqID 548 2376GGCgagggagttCTT SeqID 549 2376 AGG^(m)cgagggagttCTT SeqID 550 2377GCGagggagttCT SeqID 551 2377 GGCgagggagtTCT SeqID 552 2377AGG^(m)cgagggagtTCT SeqID 553 2377 GAGg^(m)cgagggagtTCT SeqID 554 2378GGCgagggagtTC SeqID 555 2378 AGG^(m)cgagggagTTC SeqID 556 2378GAGg^(m)cgagggagTTC SeqID 557 2378 CGAgg^(m)cgagggagTTC SeqID 558 2379AGG^(m)cgagggagTT SeqID 559 2379 GAGg^(m)cgagggaGTT SeqID 560 2379CGAgg^(m)cgagggaGTT SeqID 561 2379 GCGagg^(m)cgagggaGTT SeqID 562 2380GAGg^(m)cgagggaGT SeqID 563 2380 CGAgg^(m)cgagggAGT SeqID 564 2380GCGagg^(m)cgagggAGT SeqID 565 2380 TGCgagg^(m)cgagggAGT SeqID 566 2381CGAgg^(m)cgagggAG SeqID 567 2381 GCGagg^(m)cgaggGAG SeqID 568 2381TGCgagg^(m)cgaggGAG SeqID 569 2381 CTG^(m)cgagg^(m)cgaggGAG SeqID 5702382 CGagg^(m)cgaggGA SeqID 571 2382 GCGagg^(m)cgaggGA SeqID 572 2382TGCgagg^(m)cgagGGA SeqID 573 2382 CTG^(m)cgagg^(m)cgagGGA SeqID 574 2382TCTg^(m)cgagg^(m)cgagGGA SeqID 575 2383 TCTg^(m)cgagg^(m)cgaGGGSeqID 576 2383 GTCtg^(m)cgagg^(m)cgaGGG SeqID 577 2824 GTTcccaagaaTATSeqID 578 2824 TGTtcccaagaaTAT SeqID 579 2825 GTTcccaagaaTA SeqID 5802825 TGTtcccaagaATA SeqID 581 2825 TTGttcccaagaATA SeqID 582 2826TGTtcccaagaAT SeqID 583 2826 TTGttcccaagAAT SeqID 584 x  414GAGGcatagcagCAGG SeqID 585  691 GAAccactgaaCAAA SeqID 586  691GAACcactgaacAAA SeqID 587  691 CGaaccactgaaCAAA SeqID 588  691CGAAccactgaacAAA SeqID 589  691 CGAaccactgaaCAAA SeqID 590  691CGAAccactgaacaAA SeqID 591  691 CGAAccactgaaCAAA SeqID 592  692CGAAccactgaacAA SeqID 593  692 CGAAccactgaaCAA SeqID 594  692CGAaccactgaACAA SeqID 595  693 CGaaccactgAACA SeqID 596  693CGAAccactgaaCA SeqID 597  693 CGAaccactgAACA SeqID 598  693CGAAccactgaACA SeqID 599  694 CGaaccactgAAC SeqID 600  694 CGAaccactgAACSeqID 601 1264 CCgcagtatggATCG SeqID 602 1264 CCGCagtatggatCG SeqID 6031264 CCGCagtatggaTCG SeqID 604 1264 CCGcagtatggATCG SeqID 605 1265CGCAgtatggaTC SeqID 606 1265 CGcagtatggATC SeqID 607 1265 CGCagtatggATCSeqID 608 1265 CGcagtatgGATC SeqID 609 1530 GCGTaaagagagGT SeqID 6101530 GCgtaaagagAGGT SeqID 611 1530 GCGtaaagagAGGT SeqID 612 1530GCGTaaagagaGGT SeqID 613 1530 CGCGtaaagagagGT SeqID 614 1530CGcgtaaagagAGGT SeqID 615 1530 CGCGtaaagagaGGT SeqID 616 1530CGCgtaaagagAGGT SeqID 617 1531 GCgtaaagagAGG SeqID 618 1531GCGtaaagagAGG SeqID 619 1531 GCgtaaagaGAGG SeqID 620 1531 GCGTaaagagaGGSeqID 621 1551 AGaaggcacagACGG SeqID 622 1551 AGAaggcacagACGG SeqID 6231551 AGAAggcacagaCGG SeqID 624 1551 GAGAaggcacagaCGG SeqID 625 1551GAGaaggcacagACGG SeqID 626 1551 GAGAaggcacagACGG SeqID 627 1577GAagtgcacaCGG SeqID 628 1577 GAAgtgcacaCGG SeqID 629 1577 GAAGtgcacaCGGSeqID 630 1577 GAAgtgcacACGG SeqID 631 1577 GCgaagtgcacaCGG SeqID 6321577 GCGaagtgcacacGG SeqID 633 1577 GCGAagtgcacacGG SeqID 634 1577GCgaagtgcacACGG SeqID 635 1577 AGCGaagtgcacacGG SeqID 636 1577AG^(m)cgaagtgcacACGG SeqID 637 1577 AG^(m)cgaagtgcacaCGG SeqID 638 1577AGCgaagtgcacacGG SeqID 639 1578 CGaagtgcaCACG SeqID 640 1578CGAagtgcacACG SeqID 641 1578 CGaagtgcacACG SeqID 642 1578AGCgaagtgcaCACG SeqID 643 1578 AGCGaagtgcacACG SeqID 644 1578AGCGaagtgcacaCG SeqID 645 1578 AG^(m)cgaagtgcaCACG SeqID 646 1578AAg^(m)cgaagtgcaCACG SeqID 647 1578 AAGCgaagtgcacaCG SeqID 648 1578AAG^(m)cgaagtgcaCACG SeqID 649 1578 AAGCgaagtgcacACG SeqID 650 1578AAGCgaagtgcaCACG SeqID 651 1580 GAag^(m)cgaagtgCACA SeqID 652 1580GAAG^(m)cgaagtgcaCA SeqID 653 1580 GAAg^(m)cgaagtgCACA SeqID 654 1580GAAG^(m)cgaagtgCACA SeqID 655 1582 GGtgaag^(m)cgaagtGCA SeqID 656 1582GGTgaag^(m)cgaagtgCA SeqID 657 1582 GGTGaag^(m)cgaagtgCA SeqID 658 1582GGtgaag^(m)cgaagTGCA SeqID 659 1583 GGtgaag^(m)cgaagTGC SeqID 660 1583GGTgaag^(m)cgaagtGC SeqID 661 1583 GGTGaag^(m)cgaagtGC SeqID 662 1583GGtgaag^(m)cgaaGTGC SeqID 663 1583 AGgtgaag^(m)cgaagTGC SeqID 664 1583AGGtgaag^(m)cgaagtGC SeqID 665 1583 AGGTgaag^(m)cgaagtGC SeqID 666 1583AGgtgaag^(m)cgaaGTGC SeqID 667 1584 AGGTgaag^(m)cgaagTG SeqID 668 1584AGgtgaag^(m)cgaAGTG SeqID 669 1584 AGGtgaag^(m)cgaAGTG SeqID 670 1584AGGTgaag^(m)cgaaGTG SeqID 671 1585 AGGTgaag^(m)cgaaGT SeqID 672 1585AGgtgaag^(m)cgAAGT SeqID 673 1585 AGGtgaag^(m)cgAAGT SeqID 674 1585AGGTgaag^(m)cgaAGT SeqID 675 1588 CAGAggtgaagcGA SeqID 676 1588CAgaggtgaaGCGA SeqID 677 1588 CAGaggtgaaGCGA SeqID 678  670TAGtaaactgagCCA SeqID 679  670 TAgtaaactgaGCCA SeqID 680  670TAGTaaactgagcCA SeqID 681  670 TAGtaaactgaGCCA SeqID 682  670TAGTaaactgagCCA SeqID 683  670 CTAgtaaactgagCCA SeqID 684  670CTagtaaactgaGCCA SeqID 685  670 CTAGtaaactgagcCA SeqID 686  671CTAgtaaactgaGCC SeqID 687  671 CTagtaaactgAGCC SeqID 688  671CTAGtaaactgagCC SeqID 689  671 CTagtaaactgaGCC SeqID 690  671CTAgtaaactgagCC SeqID 691  674 GCActagtaaacTGA SeqID 692  674GCactagtaaaCTGA SeqID 693  674 GCACtagtaaactGA SeqID 694  674GCActagtaaaCTGA SeqID 695  674 GCACtagtaaacTGA SeqID 696  674GGCactagtaaacTGA SeqID 697  674 GGcactagtaaaCTGA SeqID 698  674GGCActagtaaactGA SeqID 699 1141 CAA^(m)cggggtaaaGGT SeqID 700 1141CAa^(m)cggggtaaAGGT SeqID 701 1141 CAACggggtaaagGT SeqID 702 1141CAA^(m)cggggtaaAGGT SeqID 703 1141 CAACggggtaaaGGT SeqID 704 1261CAGtatggat^(m)cgGCA SeqID 705 1261 CAgtatggat^(m)cGGCA SeqID 706 1261CAgtatggat^(m)cgGCA SeqID 707 1261 CAGtatggat^(m)cggCA SeqID 708 1265TTC^(m)cgcagtatggATC SeqID 709 1265 TTc^(m)cgcagtatgGATC SeqID 710 1265TTCCgcagtatggaTC SeqID 711 1265 TTC^(m)cgcagtatgGATC SeqID 712 1265TTCCgcagtatggATC SeqID 713 1266 TTC^(m)cgcagtatgGAT SeqID 714 1266TTc^(m)cgcagtatGGAT SeqID 715 1266 TTCCgcagtatggAT SeqID 716 1266TTC^(m)cgcagtatGGAT SeqID 717 1266 TTCCgcagtatgGAT SeqID 718 1266GTTc^(m)cgcagtatgGAT SeqID 719 1266 GTtc^(m)cgcagtatGGAT SeqID 720 1266GTTC^(m)cgcagtatggAT SeqID 721 1267 GTtc^(m)cgcagtaTGGA SeqID 722 1267GTTC^(m)cgcagtatgGA SeqID 723 1267 GTtc^(m)cgcagtatGGA SeqID 724 1267GTTc^(m)cgcagtatgGA SeqID 725 1267 AGTtc^(m)cgcagtatGGA SeqID 726 1267AGTTc^(m)cgcagtatgGA SeqID 727 1267 AGttc^(m)cgcagtatGGA SeqID 728 1267AGTtc^(m)cgcagtatgGA SeqID 729 1267 AGttc^(m)cgcagtatgGA SeqID 730 1268AGTtc^(m)cgcagtaTGG SeqID 731 1268 AGttc^(m)cgcagtaTGG SeqID 732 1268AGTtc^(m)cgcagtatGG SeqID 733 1268 AGttc^(m)cgcagtatGG SeqID 734 1268GAgttc^(m)cgcagtaTGG SeqID 735 1268 GAGttc^(m)cgcagtatGG SeqID 736 1268GAgttc^(m)cgcagtatGG SeqID 737 1269 GAGTtc^(m)cgcagtaTG SeqID 738 1269GAgttc^(m)cgcagtATG SeqID 739 1269 GAGttc^(m)cgcagtaTG SeqID 740 1269GAgttc^(m)cgcagtaTG SeqID 741 1269 GGAGttc^(m)cgcagtaTG SeqID 742 1269GGagttc^(m)cgcagtATG SeqID 743 1269 GGAgttc^(m)cgcagtaTG SeqID 744 1269GGagttc^(m)cgcagtaTG SeqID 745 1525 TAAagagaggtg^(m)cGCC SeqID 746 1525TAaagagaggtgCGCC SeqID 747 1525 TAAAgagaggtc^(m)cgCC SeqID 748 1525TAAagagaggtgCGCC SeqID 749 1525 TAAAgagaggtc^(m)cGCC SeqID 750 1526TAAagagaggtgCGC SeqID 751 1526 TAaagagaggtGCGC SeqID 752 1526TAAagagaggtGCGC SeqID 753 1526 TAAAgagaggtgCGC SeqID 754 1526GTAaagagaggtgCGC SeqID 755 1526 GTaaagagaggtGCGC SeqID 756 1527GTAaagagaggtGCG SeqID 757 1527 GTaaagagaggTGCG SeqID 758 1527GTAaagagaggTGCG SeqID 759 1527 GTAAagagaggtGCG SeqID 760 1527CGtaaagagaggTGCG SeqID 761 1527 CGTAaagagaggtgCG SeqID 762 1527CGTaaagagaggTGCG SeqID 763 1527 CGTAaagagaggtGCG SeqID 764 1528CGTaaagagaggTGC SeqID 765 1528 CGtaaagagagGTGC SeqID 766 1528CGTAaagagaggtGC SeqID 767 1528 CGTaaagagagGTGC SeqID 768 1528CGTAaagagaggTGC SeqID 769 1528 GCGtaaagagaggTGC SeqID 770 1528GCgtaaagagagGTGC SeqID 771 1528 GCgtaaagagaggTGC SeqID 772 1528GCGtaaagagaggtGC SeqID 773 1529 GCGtaaagagagGTG SeqID 774 1529GCgtaaagagaGGTG SeqID 775 1529 GCGTaaagagaggTG SeqID 776 1529GCGtaaagagaGGTG SeqID 777 1529 GCGTaaagagagGTG SeqID 778 1529CGCgtaaagagagGTG SeqID 779 1529 ^(m)cg^(m)cgtaaagagaGGTG SeqID 780 1529CGCGtaaagagaggTG SeqID 781 1529 CGCgtaaagagaGGTG SeqID 782 1529CGCGtaaagagagGTG SeqID 783 1552 TGAgaaggcacagACG SeqID 784 1552TGagaaggcacaGACG SeqID 785 1552 TGAGaaggcacagaCG SeqID 786 1552TGAgaaggcacaGACG SeqID 787 1552 TGAGaaggcacagACG SeqID 788 1690GCctcaaggt^(m)cgGTC SeqID 789 1690 GCCtcaaggt^(m)cggTC SeqID 790 1690GCctcaaggt^(m)cggTC SeqID 791 1778 ATgcctacagccTCC SeqID 792 1778ATGcctacagcctCC SeqID 793 1778 ATgcctacagcctCC SeqID 794 1785ACCAatttatgcCTAC SeqID 795 1785 ACCaatttatgcCTAC SeqID 796 1785ACCAatttatgccTAC SeqID 797 1785 ACCaatttatgccTAC SeqID 798 1785ACcaatttatgcCTAC

TABLE 4  LNA oligomers with a GalNAc2 conjugate moiety linked via a C6amino linker and a cleavable ca phosphodiester linkage to theoligomer. The GalNAc2 conjugate moiety can also be substitutedwith other GalNAc conjugate moieties or sterol moieties. SEQ ID NoDesign SeqID 799 5′-GN2-C6 ca

c_(s)c_(s)a_(s)c_(s)t_(s)g_(s)a_(s)a_(s)c_(s)

-3′ SeqID 800 5′-GN2-C6 ca

a_(s)c_(s)c_(s)a_(s)c_(s)t_(s)g_(s)a_(s)a_(s)c_(s)

-3′ SeqID 801 5′-GN2-C6 ca

a_(s)c_(s)c_(s)a_(s)c_(s)t_(s)g_(s)a_(s)a_(s)

-3′ SeqID 802 5′-GN2-C6 ca

c_(s)a_(s)g_(s)t_(s)a_(s)t_(s)g_(s)g_(s)a_(s)

-3′ SeqID 803 5′-GN2-C6 ca

g_(s)t_(s)a_(s)a_(s)a_(s)g_(s)a_(s)g_(s)a_(s)

-3′ SeqID 804 5′-GN2-C6 ca

a_(s)g_(s)g_(s)c_(s)a_(s)c_(s)a_(s)g_(s)a_(s)

-3′ SeqID 805 5′-GN2-C6 ca

a_(s)a_(s)g_(s)g_(s)c_(s)a_(s)c_(s)a_(s)g_(s)a_(s)

-3′ SeqID 806 5′-GN2-C6 ca

a_(s)a_(s)g_(s)t_(s)g_(s)c_(s)a_(s)c_(s)a_(s)

-3′ SeqID 807 5′-GN2-C6 ca

g_(s)a_(s)a_(s)g_(s)t_(s)g_(s)c_(s)a_(s)c_(s)a_(s)

-3′ SeqID 808 5′-GN2-C6 ca

g_(s)a_(s)a_(s)g_(s)t_(s)g_(s)c_(s)a_(s)c_(s)

-3′ SeqID 809 5′-GN2-C6 ca

t_(s)g_(s)a_(s)a_(s)g_(s) ^(m)c_(s)g_(s)a_(s)a_(s)g_(s)

-3′ SeqID 810 5′-GN2-C6 ca

t_(s)g_(s)a_(s)a_(s)g_(s) ^(m)c_(s)g_(s)a_(s)a_(s)

-3′ SeqID 811 5′-GN2-C6 ca

a_(s)c_(s)c_(s)a_(s)c_(s)t_(s)g_(s)a_(s)

-3′ SeqID 812 5′-GN2-C6 ca

a_(s)c_(s)c_(s)a_(s)c_(s)t_(s)g_(s)a_(s)

-3′ SeqID 813 5′-GN2-C6 ca

a_(s)g_(s)t_(s)a_(s)t_(s)g_(s)g_(s)a_(s)

-3′ SeqID 814 5′-GN2-C6 ca

t_(s)a_(s)a_(s)a_(s)g_(s)a_(s)g_(s)a_(s)

-3′ SeqID 815 5′-GN2-C6 ca

t_(s)a_(s)a_(s)a_(s)g_(s)a_(s)g_(s)a_(s)

-3′ SeqID 816 5′-GN2-C6 ca

g_(s)t_(s)g_(s)c_(s)a_(s)c_(s)a_(s) ^(m)c_(s)

-3′ SeqID 817 5′-GN2-C6 ca

a_(s)g_(s)t_(s)g_(s)c_(s)a_(s)c_(s)a_(s)

-3′ SeqID 818 5′-GN2-C6 ca

t_(s)g_(s)a_(s)a_(s)g_(s) ^(m)c_(s)g_(s)a_(s)

-3′ SeqID 819 5′-GN2-C6 ca

_(s)c_(s)c_(s)a_(s)c_(s)t_(s)g_(s)a_(s)a_(s)

-3′ SeqID 820 5′-GN2-C6 ca^(m)

_(s)c_(s)c_(s)a_(s)c_(s)t_(s)g_(s)a_(s)a_(s)c_(s)

-3′ SeqID 821 5′-GN2-C6 ca

_(s)g_(s)t_(s)g_(s)c_(s)a_(s)c_(s)a_(s)

-3′ SeqID 822 5′-GN2-C6 ca

_(s)t_(s)a_(s)a_(s)a_(s)c_(s)t_(s)g_(s)a_(s)g_(s)

_(s)3′ SeqID 823 5′-GN2-C6 ca

_(s)a_(s)c_(s)c_(s)a_(s)c_(s)t_(s)g_(s)

-3′ SeqID 824 5′-GN2-C6 ca

_(s)a_(s)c_(s)c_(s)a_(s)c_(s)t_(s)g_(s)

-3′ SeqID 825 5′-GN2-C6 ca

C _(s) G _(s)t_(s)a_(s)a_(s)a_(s)g_(s)a_(s)g_(s)

-3′ SeqID 826 5′-GN2-C6 ca

_(s)g_(s)t_(s)g_(s)a_(s)a_(s)g_(s) ^(m)c_(s)g_(s)a_(s)

-3′ The oligomer sequence motif which these oligomers are based on is asubset from Table 2 and 3. Upper case letters denote beta-D-oxy LNA,lower case letters denote DNA, ^(m)c/^(m)C denotes a 5-methylcytosineDNA/LNA, s denotes phosphorothiate internucleoside linkages. Wherenothing is specified the linkage is a phosphodiester internucleosidelinkage

Prior to conjugation with GalNAc, the LNA oligomers of table 4 arerepresented as AM-C6 ca-oligomer, where AM-C6 represents an amino linkerready for conjugation and ca is a cleavable phosphodiester linkage.These oligomers are incorporated by reference from table 4 in thepriority application GB1408623.5.

Embodiments

The following embodiments of the present invention, presented asnumbered paragraphs, may be used in combination with the otherembodiments described herein:

-   1. An oligomer conjugate for use in the treatment of a viral    disorder, wherein said oligomer conjugate comprises:    -   a) at least one first oligomer region capable of modulating a        target sequence of Hepatitis B Virus (HBV), preferably HBx or        HBsAg of HBV, to treat said viral disorder; and    -   b) a carrier component for delivering said first oligomer to the        liver.-   2. The oligomer conjugate for the use according to paragraph 1    wherein said carrier component is capable of delivering said    oligomer to the liver of a subject to be treated by administration    of said oligomer conjugate.-   3. The oligomer conjugate for the use according to paragraph 1 or    paragraph 2 wherein said carrier component is capable of delivering    said oligomer to a hepatocyte of a subject to be treated by    administration of said oligomer conjugate.-   4. The oligomer conjugate for the use according to any of paragraphs    1 to 3 wherein said carrier component is a carbohydrate conjugate    moiety.-   5. The oligomer conjugate for the use according to any of paragraphs    1 to 4 wherein said carrier component is an asialoglycoprotein    receptor (ASGP-R) targeting moiety.-   6. The oligomer conjugate for the use according to paragraphs 4 or 5    wherein said carbohydrate conjugate moiety or ASGP-R targeting    moiety is selected from the group consisting of galactose,    galactosamine, N-formyl-galactosamine, N-acetylgalactosamine    (GalNAc), N-propionyl-galactosamine, N-n-butanoyl-galactosamine,    N-isobutanoylgalactose-amine or a cluster of any one or more    thereof.-   7. The oligomer conjugate for the use according to any of paragraphs    1 to 6 wherein said carrier component is a GalNAc cluster comprising    two to four terminal galactose derivatives, a hydrophilic spacer    linking each galactose derivative to a branch point group.-   8. The oligomer conjugate for the use according to paragraph 7,    wherein the galactose derivatives are GalNAc, the spacer is a PEG    spacer and the branch point group is a comprising a peptide, with    two or more amino groups, such as a di-lysine or tri-lysine.-   9. The oligomer conjugate for the use according to any of paragraphs    1 to 8 wherein said carrier component is GalNAc2.-   10. The oligomer conjugate for the use according to any of    paragraphs 1 to 9 wherein said first oligomer region has at least    80% complementarity to the target sequence.-   11. The oligomer conjugate for the use according to any of    paragraphs 1 to 10 wherein said first oligomer region comprises one    or more LNA units.-   12. The oligomer conjugate for the use according to any of    paragraphs 1 to 11 wherein said first oligomer region is a gapmer.-   13. The oligomer conjugate for the use according to any of    paragraphs 1 to 12 wherein said first oligomer region comprises a    2′-deoxyribonucleotide gap region flanked on each side by a wing,    wherein each wing independently comprises one or more LNA units.-   14. The oligomer conjugate for the use according to any of    paragraphs 1 to 13 wherein said first oligomer region comprises any    one of the motifs: 2-8-2, 3-8-3, 2-8-3, 3-8-2, 2-9-2, 3-9-3, 2-9-3,    3-9-2, 2-10-2, 3-10-3, 3-10-2, 2-10-3 wherein the first number is    the number of LNA units in the 5′ LNA wing region, the second number    is the number of nucleotides in the gap region, and the third number    is the number of LNA units in the 3′ LNA wing region.-   15. The oligomer conjugate for the use according to any of paragraph    1 to 14 wherein said first oligomer region is 8-30 nucleotides in    length.-   16. The oligomer conjugate for the use according to any of    paragraphs 1 to 15 wherein said first oligomer region is 10-20    nucleotides in length.-   17. The oligomer conjugate for the use according to any of    paragraphs 1 to 16 wherein said first oligomer region is 10 to 16    nucleotides in length.-   18. The oligomer conjugate for the use according to any of    paragraphs 1 to 17 wherein said first oligomer region is 10 to 14    nucleotides in length.-   19. The oligomer conjugate for the use according to any of    paragraphs 1 to 18 wherein said first oligomer region binds to the    target sequence.-   20. The oligomer conjugate for the use according to any of    paragraphs 1 to 19 wherein said first oligomer region is capable of    inhibiting any one or more of the expression, replication or    translation of the target sequence.-   21. The oligomer conjugate for the use according to any of    paragraphs 1 to 20 wherein said first oligomer region is capable of    inhibiting the expression of the target sequence.-   22. The oligomer conjugate for the use according to any of    paragraphs 1 to 21 wherein said target sequence is a gene or a mRNA.-   23. The oligomer conjugate for the use according to any of    paragraphs 1 to 22 wherein said target sequence comprises at least    part of a gene or a mRNA encoding HBx or HBsAg or a    naturally-occurring variant thereof.-   24. The oligomer conjugate for the use according to any of    paragraphs 1 to 23 wherein said target sequence is a gene or a mRNA    encoding HBx or HBsAg or a naturally-occurring variant thereof.-   25. The oligomer conjugate for the use according to any of    paragraphs 1 to 24 wherein said target sequence is within the    sequence shown as SEQ ID No. 1 and/or SEQ ID No. 2, or a sequence    that has at least 80% identity thereto, preferably at least 85%    identity thereto, preferably at least 90% identity thereto,    preferably at least 95% identity thereto.-   26. The oligomer conjugate for the use according to any of    paragraphs 1 to 24 wherein said target sequence is within the    sequence shown as SEQ ID No. 1 and SEQ ID No. 2.-   27. The oligomer conjugate for the use according to any of    paragraphs 1 to 26 wherein said target sequence has at least 80%, at    least 85%, at least 90%, at least 95%, at least 98% or at least 99%    identity with any one or more of the HBV genotypes A-H.-   28. The oligomer conjugate for the use according to any of    paragraphs 1 to 27 wherein said target sequence is selected from the    group consisting of any one or more of positions:    -   1264-1278;    -   1530-1544;    -   1551-1566;    -   1577 to 1598;    -   691-706;    -   670-684    -   of SEQ ID NO: 3.-   29. The oligomer conjugate for the use according to any of    paragraphs 1 to 28 wherein said first oligomer region is based on a    core motif selected from the group consisting of any one or more of:

(SEQ ID NO: 13) GCGTAAAGAGAGG; (SEQ ID NO: 11) GCGTAAAGAGAGGT;(SEQ ID NO: 20) AGCGAAGTGCACACG; (SEQ ID NO: 26) AGGTGAAGCGAAGTG;(SEQ ID NO 18) AGCGAAGTGCACACGG; (SEQ ID NO: 7) CGAACCACTGAACA;(SEQ ID NO 4) GAACCACTGAACAAA; (SEQ ID NO 5) CGAACCACTGAACAAA;(SEQ ID NO 6) CGAACCACTGAACAA; (SEQ ID NO 8) CGAACCACTGAAC (SEQ ID NO 9)CCGCAGTATGGATCG (SEQ ID NO: 10) CGCAGTATGGATC; (SEQ ID NO 12)CGCGTAAAGAGAGGT; (SEQ ID NO 14) AGAAGGCACAGACGG; (SEQ ID NO 15)GAGAAGGCACAGACGG (SEQ ID NO 16) GAAGTGCACACGG; (SEQ ID NO 17)GCGAAGTGCACACGG; (SEQ ID NO 19) CGAAGTGCACACG; (SEQ ID NO 27)AGGTGAAGCGAAGT; and (SEQ ID NO: 852) TAGTAAACTGAGCCA.

-   30. The oligomer conjugate for the use according any of paragraphs 1    to 29 wherein said first oligomer region is based on a sequence    selected from the group consisting of any one or more of:

(SEQ ID NO: 303) GCGtaaagagaGG; (SEQ ID NO: 301) GCGtaaagagaGGT;(SEQ ID NO: 618) GCGtaaagagAGG; (SEQ ID NO: 310) AGCgaagtgcacACG(SEQ ID NO: 668) AGgtgaagcgaAGTG; (SEQ ID NO: 308) AGCgaagtgcacaCGG;(SEQ ID NO: 297) CGAaccactgaACA; (SEQ ID NO: 300) CGCagtatggaTC;(SEQ ID NO: 315) AGGtgaagcgaagTGC; (SEQ ID NO: 316) AGGtgaagcgaaGTG;(SEQ ID NO: 294) GAAccactgaacAAA; (SEQ ID NO: 295) CGAaccactgaacAAA;(SEQ ID NO: 296) CGAaccactgaaCAA; (SEQ ID NO: 298) CGAaccactgaAC;(SEQ ID NO: 299) CCGcagtatggaTCG; (SEQ ID NO: 302) CGCgtaaagagaGGT;(SEQ ID NO: 304) AGAaggcacagaCGG; (SEQ ID NO: 305) GAGaaggcacagaCGG;(SEQ ID NO: 306) GAAgtgcacacGG; (SEQ ID NO: 307) GCGaagtgcacaCGG;(SEQ ID NO: 309) CGAagtgcacaCG; (SEQ ID NO: 585) GAAccactgaaCAAA;(SEQ ID NO: 588) CGAAccactgaacAAA (SEQ ID NO: 628) GAAgtgcacaCGG;(SEQ ID NO: 678) TAGtaaactgagCCA; (SEQ ID NO: 600) CGAaccactgAAC;(SEQ ID NO: 317) AGGtgaagcgaAGT; and (SEQ ID NO: 597) CGAaccactgAACA.

-   -   wherein uppercase letters denote LNA units and lower case        letters denote DNA units.

-   31. The oligomer conjugate for the use according to any of    paragraphs 1 to 30 wherein said oligomer conjugate is selected from    the group consisting of any one or more of:

(SEQ ID NO: 815) 5′-GN2-C6 ca

t_(s)a_(s)a_(s)a_(s)g_(s)a_(s)g_(s)a_(s)

-3′ (SEQ ID NO: 814) 5′-GN2-C6 ca

t_(s)a_(s)a_(s)a_(s)g_(s)a_(s)g_(s)a_(s)

-3′ (SEQ ID NO: 825) 5′-GN2-C6 ca

_(s)t_(s)a_(s)a_(s)a_(s)g_(s)a_(s)g_(s)

-3′ (SEQ ID NO: 808) 5′-GN2-C6 ca

g_(s)a_(s)a_(s)g_(s)t_(s)g_(s)c_(s)a_(s)c_(s)

-3′ (SEQ ID NO: 826) 5′-GN2-C6 ca

_(s)g_(s)t_(s)g_(s)a_(s)a_(s)g_(s) ^(m)c_(s)g_(s)a_(s)

-3′ (SEQ ID NO: 807) 5′-GN2-C6 ca

g_(s)a_(s)a_(s)g_(s)t_(s)g_(s)c_(s)a_(s)c_(s)a_(s)

-3′ (SEQ ID NO: 799) 5′-GN2-C6 ca

c_(s)c_(s)a_(s)c_(s)t_(s)g_(s)a_(s)a_(s)c_(s)

-3′ (SEQ ID NO: 800) 5′-GN2-C6 ca

a_(s)c_(s)c_(s)a_(s)cst_(s)g_(s)a_(s)a_(s)c_(s)

-3′ (SEQ ID NO: 801) 5′-GN2-C6 ca

a_(s)c_(s)c_(s)a_(s)c_(s)t_(s)g_(s)a_(s)a_(s)

-3′ (SEQ ID NO: 802) 5′-GN2-C6 ca

c_(s)a_(s)g_(s)t_(s)a_(s)t_(s)g_(s)g_(s)a_(s)

-3′ (SEQ ID NO: 803) 5′-GN2-C6 ca

g_(s)t_(s)a_(s)a_(s)a_(s)g_(s)a_(s)g_(s)a_(s)

-3′ (SEQ ID NO: 804) 5′-GN2-C6 ca

a_(s)g_(s)g_(s)c_(s)a_(s)c_(s)a_(s)g_(s)a_(s)

-3′ (SEQ ID NO: 805) 5′-GN2-C6 ca

a_(s)a_(s)g_(s)g_(s)c_(s)a_(s)c_(s)a_(s)g_(s)a

-3′ (SEQ ID NO: 806) 5′-GN2-C6 caG

a_(s)a_(s)g_(s)t_(s)g_(s)c_(s)a_(s)c_(s)a_(s)

-3′ (SEQ ID NO: 809) 5′-GN2-C6 ca

t_(s)g_(s)a_(s)a_(s)g_(s) ^(m)c_(s)g_(s)a_(s)a_(s)g_(s)

-3′ (SEQ ID NO: 810) 5′-GN2-C6 ca

t_(s)g_(s)a_(s)a_(s)g_(s) ^(m)c_(s)g_(s)a_(s)a_(s)

-3′ (SEQ ID NO: 811) 5′-GN2-C6 ca

a_(s)c_(s)c_(s)a_(s)c_(s)t_(s)c_(s)g_(s)a_(s)

-3′ (SEQ ID NO: 812) 5′-GN2-C6 c

a_(s)c_(s)c_(s)a_(s)c_(s)t_(s)g_(s)a_(s)

-3′ (SEQ ID NO: 813) 5′-GN2-C6 ca

a_(s)g_(s)t_(s)a_(s)t_(s)g_(s)g_(s)a_(s)

-3 (SEQ ID NO: 816) 5′-GN2-C6 ca

g_(s)t_(s)g_(s)c_(s)a_(s)c_(s)a_(s) ^(m)c_(s)

-3′ (SEQ ID NO: 817) 5′-GN2-C6 c

a_(s)g_(s)t_(s)g_(s)c_(s)a_(s)c_(s)a_(s)

-3′ (SEQ ID NO: 818) 5′-GN2-C6 ca

t_(s)g_(s)a_(s)a_(s)g_(s) ^(m)c_(s)g_(s)a_(s)

-3′ (SEQ ID NO: 819) 5′-GN2-C6 ca

_(s)c_(s)c_(s)a_(s)c_(s)t_(s)g_(s)a_(s)a_(s)

-3′ (SEQ ID NO: 820) 5′-GN2-C6 ca^(m)

_(s)c_(s)c_(s)a_(s)c_(s)t_(s)g_(s)a_(s)a_(s)c_(s)

-3′ (SEQ ID NO: 821) 5′-GN2-C6 ca

g_(s)t_(s)g_(s)c_(s)a_(s)c_(s)a_(s)

-3′ (SEQ ID NO: 822) 5′-GN2-C6 ca

_(s)t_(s)a_(s)a_(s)a_(s)c_(s)t_(s)g_(s)a_(s)g_(s)

3′ (SEQ ID NO: 823) 5′-GN2-C6 ca

_(s)a_(s)c_(s)c_(s)a_(s)c_(s)t_(s)g_(s)

-3′ (SEQ ID NO: 824) 5′-GN2-C6 ca

_(s)a_(s)c_(s)c_(s)a_(s)c_(s)t_(s)g_(s)

-3′

-   -   wherein uppercase letters denote beta-D-oxy-LNA units; lowercase        letters denote DNA units; the subscript “s” denotes a        phosphorothioate linkage; superscript m denotes a DNA or        beta-D-oxy-LNA unit containing a 5-methylcytosine base; GN2-C6        denotes a GalNAc2 carrier component with a C6 linker.

-   32. The oligomer conjugate for the use according to any of    paragraphs 1 to 29 wherein said first oligomer region is based on a    core motif selected from the group consisting of any one or more of:

(SEQ ID NO: 13) GCGTAAAGAGAGG (SEQ ID NO: 11) GCGTAAAGAGAGGT and(SEQ ID NO 12) CGCGTAAAGAGAGGT.

-   33. The oligomer conjugate for the use according to any of    paragraphs 1 to 29 wherein said first oligomer region is based on a    core motif selected from the group consisting of any one or more of:

(SEQ ID NO: 20) AGCGAAGTGCACACG; (SEQ ID NO: 26) AGGTGAAGCGAAGTG;(SEQ ID NO 18) AGCGAAGTGCACACGG; (SEQ ID NO 16) GAAGTGCACACGG;(SEQ ID NO 17) GCGAAGTGCACACGG; (SEQ ID NO 19) CGAAGTGCACACG and(SEQ ID NO 27) AGGTGAAGCGAAGT.

-   34. The oligomer conjugate for the use according to any one of    paragraphs 1 to 33 wherein said oligomer conjugate has or comprises    the structure:    -   Carrier component-L1-First Oligomer Region    -   wherein L1 is an optional linker or brancher region or tether        molecule or bridging moiety; or    -   wherein said oligomer conjugate has or comprises the structure:    -   First Oligomer Region-L2-Carrier component    -   wherein L2 is an optional linker or brancher region or tether        molecule or bridging moiety.-   35. The oligomer conjugate for the use according to any one of    paragraphs 1 to 34 wherein said oligomer conjugate has or comprises    the structure:    -   Carrier component-L1-First Oligomer Region    -   wherein L1 is an optional linker.-   36. The oligomer conjugate for the use according to paragraph 34 or    paragraph 35 wherein said Linker 1 is present.-   37. The oligomer conjugate for the use according to any of    paragraphs 1 to 36 wherein said carrier component is linked,    preferably conjugated, to the 5′ end of said oligomer.-   38. The oligomer conjugate for the use according to paragraph 34 to    37 wherein the linker group or the brancher region is a    physiologically labile linker group or a physiologically labile    brancher region or physiologically labile tether molecule or    physiologically labile bridging moiety.-   39. The oligomer conjugate for the use according to paragraph 38    wherein the physiologically labile linker group is a nuclease    susceptible linker.-   40. The oligomer conjugate for the use according to paragraph 38 or    39 wherein the physiologically labile linker further is conjugated    with a C6 to C12 amino alkyl group.-   41. The oligomer conjugate for the use according to any of    paragraphs 1 to 40 which further comprises a second oligomer region    which is capable of modulating a target sequence.-   42. The oligomer conjugate for the use according to paragraph 41    wherein each of the first oligomer region and the second oligomer    regions is capable of modulating a target sequence in HBx or HBsAg    of HBV.-   43. The oligomer conjugate for the use according to any one of    paragraphs 31 or 42 wherein each of the first oligomer region and    the second oligomer regions is capable of modulating a target    sequence in HBx or HBsAg of HBV; wherein said target regions are    different.-   44. The oligomer conjugate for the use according to any one of    paragraphs 41 to 43 wherein said oligomer conjugate has or comprises    the structure:    -   Carrier component-L1-First Oligomer Region-L2-Second Oligomer        Region    -   wherein L1 is an optional linker or brancher region or tether        molecule or bridging moiety    -   wherein L2 is an optional linker or brancher region or tether        molecule or bridging moiety    -   wherein L1 and L2 can be the same or different; or    -   wherein said oligomer conjugate has or comprises the structure:    -   First Oligomer Region-L2-Second Oligomer Region-L3-Carrier        component    -   wherein L2 is an optional linker or brancher region or tether        molecule or bridging moiety    -   wherein L3 is an optional linker or brancher region or tether        molecule or bridging moiety    -   wherein L2 and L3 can be the same or different; or    -   wherein said oligomer conjugate has or comprises the structure:    -   Carrier component 1-L1-First Oligomer Region-L2-Second Oligomer        Region-L3-Carrier component 2    -   wherein L1 is an optional linker or brancher region or tether        molecule or bridging moiety    -   wherein L2 is an optional linker or brancher region or tether        molecule or bridging moiety    -   wherein L3 is an optional linker or brancher region or tether        molecule or bridging moiety    -   wherein L1, L2 and L3 can be the same or different    -   wherein Carrier component 1 and Carrier component 2 can be the        same or different; or    -   wherein said oligomer conjugate has or comprises the structure:    -   First Oligomer Region-L1-Carrier component 1-L2-Second Oligomer        Region    -   wherein L1 is an optional linker or brancher region or tether        molecule or bridging moiety    -   wherein L2 is an optional linker or brancher region or tether        molecule or bridging moiety    -   wherein L1 and L2 and L3 can be the same or different; or    -   wherein said oligomer conjugate has or comprises the structure:    -   First Oligomer Region-L1-Carrier component 1-L2-Second Oligomer        Region-L3-Carrier component 2    -   wherein L1 is an optional linker or brancher region or tether        molecule or bridging moiety    -   wherein L2 is an optional linker or brancher region or tether        molecule or bridging moiety    -   wherein L3 is an optional linker or brancher region or tether        molecule or bridging moiety    -   wherein L1 and L2 can be the same or different    -   wherein Carrier component 1 and Carrier component 2 can be the        same or different; or.    -   wherein said oligomer conjugate has or comprises the structure:    -   Carrier component 1-L1-First Oligomer Region-L2-Carrier        component 2-L3-Second Oligomer Region-L4-Carrier component 3    -   wherein L1 is an optional linker or brancher region or tether        molecule or bridging moiety    -   wherein L2 is an optional linker or brancher region or tether        molecule or bridging moiety    -   wherein L3 is an optional linker or brancher region or tether        molecule or bridging moiety    -   wherein L1, L2 and L3 can be the same or different    -   wherein Carrier component 1, Carrier Component 2 and Carrier        component 3 can be the same or different.-   45. The oligomer conjugate for the use according to any one of    paragraphs 39 to 43 wherein said oligomer conjugate has or comprises    the structure:    -   Carrier component-L1-First Oligomer Region-L2-Second Oligomer        Region    -   wherein L1 is an optional linker or brancher region or tether        molecule or bridging moiety    -   wherein L2 is an optional linker or brancher region or tether        molecule or bridging moiety    -   wherein L1 and L2 can be the same or different.-   46. The oligomer conjugate for the use according to paragraph 45    wherein said Linker 1 is present.-   47. The oligomer conjugate for the use according to paragraph 45 or    paragraph 46 wherein said Linker 2 is present.-   48. The oligomer conjugate for the use according to any of    paragraphs 41 to 46 wherein said carrier component is linked,    preferably conjugated, to the 5′ end of said oligomer.-   49. The oligomer conjugate for the use according to any of    paragraphs 41 to 48 wherein each of the first oligomer region and    the second oligomer regions is linked, preferably conjugated, by    means of a linker or brancher region.-   50. The oligomer conjugate for the use according to any of    paragraphs 41 to 49 wherein each of the first oligomer region and    the second oligomer regions is linked, preferably conjugated, by    means of a physiologically labile linker group or a physiologically    labile brancher region.-   51. The oligomer conjugate for the use according to any of    paragraphs 1 to 50 wherein said viral disorder is hepatitis B or a    disorder associated with HBV-   52. The oligomer conjugate for the use according to any of    paragraphs 1 to 51 wherein said viral disorder is associated with    expression or overexpression of HBx or HBsAg.-   53. The oligomer conjugate for the use according to any of    paragraphs 1 to 52, wherein said oligomer conjugate is administered    subcutaneously.-   54. A composition for use in the treatment of a viral disorder,    wherein said composition comprises an oligomer conjugate as defined    in any one of paragraphs 1 to 53 and at least one additional    different oligonucleotide.-   55. The composition for the use according to paragraph 54 wherein at    least one of said additional different oligonucleotide is an    oligomer conjugate.-   56. The composition for the use according to paragraph 54 or    paragraph 55 wherein each of said additional different    oligonucleotide is an oligomer conjugate.-   57. The composition for the use according to any one of paragraphs    54 to 56 wherein each of said additional different oligonucleotide    is capable of modulating a target sequence in HBV.-   58. The composition for the use according to any one of paragraphs    54 to 57 wherein at least one of said additional different    oligonucleotide is capable of modulating a target sequence in HBx or    HBsAg of HBV.-   59. The composition for the use according to any one of paragraphs    54 to 58 wherein at least one of said additional different    oligonucleotide is capable of modulating a target sequence in HBx or    HBsAg of HBV; and wherein said at least one of said additional    different oligonucleotide is capable of modulating a target sequence    in HBx or HBsAg of HBV different to that targeted by an oligomer    conjugate as defined in any one of paragraphs 1 to 53.-   60. The composition for the use according to any one of paragraphs    54 to 59 wherein each of said additional different oligonucleotide    is capable of modulating a target sequence in HBx or HBsAg of HBV.-   61. The composition for the use according to any one of paragraphs    54 to 60 wherein each of said additional different oligonucleotide    is capable of modulating a target sequence in HBx or HBsAg of HBV;    and wherein each of said additional different oligonucleotide is    capable of modulating a target sequence in HBx or HBsAg of HBV    different to that targeted by an oligomer conjugate as defined in    any one of paragraphs 1 to 53.-   62. An oligomer conjugate suitable for the treatment of a viral    disorder, wherein said oligomer conjugate comprises:    -   a) at least one first oligomer region capable of modulating a        target sequence of Hepatitis B Virus (HBV), preferably HBx or        HBsAg of HBV, to treat said viral disorder; and    -   b) a carrier component for delivering said first oligomer to the        liver.-   63. An oligomer conjugate according to paragraph 62 wherein said    oligomer is as defined in any one of paragraphs 1 to 53.-   64. A composition suitable for the treatment of a viral disorder,    wherein said composition comprises an oligomer conjugate and at    least one additional different oligonucleotide; wherein said    oligomer conjugate comprises:    -   a) at least one first oligomer region capable of modulating a        target sequence of Hepatitis B Virus (HBV), preferably HBx or        HBsAg of HBV, to treat said viral disorder; and    -   b) a carrier component for delivering said first oligomer to the        liver.-   65. A composition according to paragraph 64 wherein said oligomer    conjugate is an oligomer conjugate as defined in any one of    paragraphs 1 to 53 or any one of paragraphs 62 to 63.-   66. A composition according to paragraph 64 or paragraph 65 wherein    said additional different oligonucleotide is an additional different    oligonucleotide as defined in any one of paragraphs 62 to 63.-   67. An oligomer based on a core motif selected from the group    consisting of any one or more of:

(SEQ ID NO: 13) GCGTAAAGAGAGG; (SEQ ID NO: 11) GCGTAAAGAGAGGT;(SEQ ID NO: 20) AGCGAAGTGCACACG; (SEQ ID NO: 26) AGGTGAAGCGAAGTG;(SEQ ID NO: 7) CGAACCACTGAACA; (SEQ ID NO 4) GAACCACTGAACAAA;(SEQ ID NO 5) CGAACCACTGAACAAA; (SEQ ID NO 6) CGAACCACTGAACAA;(SEQ ID NO 8) CGAACCACTGAAC (SEQ ID NO 9) CCGCAGTATGGATCG(SEQ ID NO: 10) CGCAGTATGGATC; (SEQ ID NO 12) CGCGTAAAGAGAGGT;(SEQ ID NO 14) AGAAGGCACAGACGG; (SEQ ID NO 15) GAGAAGGCACAGACGG(SEQ ID NO 16) GAAGTGCACACGG; (SEQ ID NO 17) GCGAAGTGCACACGG;(SEQ ID NO 19) CGAAGTGCACACG; (SEQ ID NO 27) AGGTGAAGCGAAGT; and(SEQ ID NO: 852) TAGTAAACTGAGCCA

-   -   which is capable of modulating a target sequence in HBx or HBsAg        of HBV to treat a viral disorder.

-   68. The oligomer of paragraph 67 wherein said oligomer region is    based on a core motif selected from the group consisting of any one    or more of:

(SEQ ID NO: 13) GCGTAAAGAGAGG (SEQ ID NO: 11) GCGTAAAGAGAGGT and(SEQ ID NO 12) CGCGTAAAGAGAGGT.

-   69. The oligomer of paragraph 67 wherein said oligomer region is    based on a core motif selected from the group consisting of any one    or more of:

(SEQ ID NO: 20) AGCGAAGTGCACACG; (SEQ ID NO: 26) AGGTGAAGCGAAGTG;(SEQ ID NO 18) AGCGAAGTGCACACGG; (SEQ ID NO 16) GAAGTGCACACGG;(SEQ ID NO 17) GCGAAGTGCACACGG; (SEQ ID NO 19) CGAAGTGCACACG and(SEQ ID NO 27) AGGTGAAGCGAAGT.

-   70. The oligomer according to any one of paragraphs 67 to 69,    wherein said oligomer comprises one or more LNA units.-   71. The oligomer according to any of paragraphs 67 to 70 wherein    said oligomer is a gapmer.-   72. The oligomer according to any of paragraphs 67 to 71 wherein    said oligomer comprises a 2′-deoxyribonucleotide gap region flanked    on each side by a wing, wherein each wing independently comprises    one or more LNA units.-   73. The oligomer according to any of paragraphs 67 to 72 wherein    said oligomer any one of the motifs: 2-8-2, 3-8-3, 2-8-3, 3-8-2,    2-9-2, 3-9-3, 2-9-3, 3-9-2, 2-10-2, 3-10-3, 3-10-2, 2-10-3 wherein    the first number is the number of LNA units in an LNA wing region,    the second number is the number of nucleotides in the gap region,    and the third number is the number of LNA units in an LNA wing    region.-   74. The oligomer according to any of paragraphs 67 to 73 wherein    said first oligomer region is 10-18 nucleotides in length.-   75. The oligomer according to any of paragraphs 67 to 74 wherein    said first oligomer region is 10 to 16 nucleotides in length.-   76. The oligomer according to any of paragraphs 67 to 75 wherein    said first oligomer region is 10 to 14 nucleotides in length.-   77. The oligomer according to any one of paragraphs 67 to 76 which    is based on a sequence selected from the group consisting of any one    or more of:

(SEQ ID NO: 303) GCGtaaagagaGG; (SEQ ID NO: 301) GCGtaaagagaGGT;(SEQ ID NO: 618) GCGtaaagagAGG; (SEQ ID NO: 310) AGCgaagtgcacACG(SEQ ID NO: 668) AGgtgaagcgaAGTG; (SEQ ID NO: 308) AGCgaagtgcacaCGG;(SEQ ID NO: 297) CGAaccactgaACA; (SEQ ID NO: 300) CGCagtatggaTC;(SEQ ID NO: 315) AGGtgaagcgaagTGC; (SEQ ID NO: 316) AGGtgaagcgaaGTG;(SEQ ID NO: 294) GAAccactgaacAAA; (SEQ ID NO: 295) CGAaccactgaacAAA;(SEQ ID NO: 296) CGAaccactgaaCAA; (SEQ ID NO: 298) CGAaccactgaAC;(SEQ ID NO: 299) CCGcagtatggaTCG; (SEQ ID NO: 302) CGCgtaaagagaGGT;(SEQ ID NO: 304) AGAaggcacagaCGG; (SEQ ID NO: 305) GAGaaggcacagaCGG;(SEQ ID NO: 306) GAAgtgcacacGG; (SEQ ID NO: 307) GCGaagtgcacaCGG;(SEQ ID NO: 309) CGAagtgcacaCG; (SEQ ID NO: 585) GAAccactgaaCAAA;(SEQ ID NO: 588) CGAAccactgaacAAA (SEQ ID NO: 628) GAAgtgcacaCGG;(SEQ ID NO: 678) TAGtaaactgagCCA; (SEQ ID NO: 600) CGAaccactgAAC;(SEQ ID NO: 317) AGGtgaagcgaAGT; and (SEQ ID NO: 597) CGAaccactgAACA,wherein uppercase letters denote affinity enhancing nucleotide analoguesand lower case letters denote DNA units.

-   78. The oligomer according to any of paragraphs 67 to 77 which is    based on a selected from a sequence selected from the group    consisting of any one or more of:

(SEQ ID NO: 303) 5′-AM-C6 ca

t_(s)a_(s)a_(s)a_(s)g_(s)a_(s)g_(s)a_(s)

-3′ (SEQ ID NO: 301) 5′-AM-C6 ca

t_(s)a_(s)a_(s)a_(s)g_(s)a_(s)g_(s)a_(s)

-3′ (SEQ ID NO: 618) 5′-AM-C6 ca

_(s)t_(s)a_(s)a_(s)a_(s)g_(s)a_(s)g_(s)

-3′ (SEQ ID NO: 310) 5′-AM-C6 ca

g_(s)a_(s)a_(s)g_(s)t_(s)g_(s)c_(s)a_(s)c_(s)

-3′ (SEQ ID NO: 668) 5′-AM-C6 ca

_(s)g_(s)t_(s)g_(s)a_(s)a_(s)g_(s) ^(m)c_(s)g_(s)a_(s)

-3′ (SEQ ID NO: 308) 5′-AM-C6 ca

g_(s)a_(s)a_(s)g_(s)t_(s)g_(s)c_(s)a_(s)c_(s)a_(s)

-3′ (SEQ ID NO: 294) 5′-AM-C6 ca

c_(s)c_(s)a_(s)c_(s)t_(s)g_(s)a_(s)a_(s)c_(s)

-3′ (SEQ ID NO: 295) 5′-AM-C6 ca

a_(s)c_(s)c_(s)a_(s)c_(s)t_(s)g_(s)a_(s)a_(s)c_(s)

-3′ (SEQ ID NO: 296) 5′-AM-C6 ca

a_(s)c_(s)c_(s)a_(s)c_(s)t_(s)g_(s)a_(s)a_(s)

-3′ (SEQ ID NO: 299) 5′-AM-C6 ca

c_(s)a_(s)g_(s)t_(s)a_(s)t_(s)g_(s)g_(s)a_(s)

-3′ (SEQ ID NO: 302) 5′-AM-C6 ca

g_(s)t_(s)a_(s)a_(s)a_(s)g_(s)a_(s)g_(s)a_(s)

-3′ (SEQ ID NO: 304) 5′-AM-C6 ca

a_(s)g_(s)g_(s)c_(s)a_(s)c_(s)a_(s)g_(s)a_(s)

-3′ (SEQ ID NO: 305) 5′-AM-C6 ca

a_(s)a_(s)g_(s)g_(s)c_(s)a_(s)c_(s)a_(s)g_(s)a_(s)

-3′ (SEQ ID NO: 307) 5′-AM-C6 ca

a_(s)a_(s)g_(s)t_(s)g_(s)c_(s)a_(s)c_(s)a_(s)

-3′ (SEQ ID NO: 315) 5′-AM-C6 ca

t_(s)g_(s)a_(s)a_(s)g_(s) ^(m)c_(s)g_(s)a_(s)a_(s)g_(s) T

-3, (SEQ ID NO: 316) 5′-AM-C6 ca

t_(s)g_(s)a_(s)a_(s)g_(s) ^(m)c_(s)g_(s)a_(s)a_(s)

-3′ (SEQ ID NO: 297) 5′-AM-C6 ca

a_(s)c_(s)c_(s)a_(s)c_(s)t_(s)g_(s)a_(s)

-3′ (SEQ ID NO: 298) 5′-AM-C6 ca

a_(s)c_(s)c_(s)a_(s)c_(s)t_(s)g_(s)a_(s)

-3′ (SEQ ID NO: 300) 5′-AM-C6 ca

a_(s)g_(s)t_(s)a_(s)t_(s)g_(s)g_(s)a_(s)

-3′ (SEQ ID NO: 306) 5′-AM-C6 ca

g_(s)t_(s)g_(s)c_(s)a_(s)c_(s)a_(s) ^(m)c_(s)

-3′ (SEQ ID NO: 309) 5′-AM-C6 ca

a_(s)g_(s)t_(s)g_(s)c_(s)a_(s)c_(s)a_(s)

-3′ (SEQ ID NO: 317) 5′-AM-C6 ca

t_(s)g_(s)a_(s)a_(s)g_(s) ^(m)c_(s)g_(s)a_(s)

-3′ (SEQ ID NO: 585) 5′-AM-C6 ca

_(s)c_(s)c_(s)a_(s)c_(s)t_(s)g_(s)a_(s)a_(s)

-3′ (SEQ ID NO: 588) 5′-AM-C6 ca^(m)

_(s)c_(s)c_(s)a_(s)c_(s)t_(s)g_(s)a_(s)a_(s)c_(s)

-3′ (SEQ ID NO: 628) 5′-AM-C6 ca

_(s)g_(s)t_(s)g_(s)c_(s)a_(s)c_(s)a_(s)

-3′ (SEQ ID NO: 678) 5′-AM-C6 ca

_(s)t_(s)a_(s)a_(s)a_(s)c_(s)t_(s)g_(s)a_(s)g_(s)

_(s)3′ (SEQ ID NO: 600) 5′-AM-C6 ca

_(s)a_(s)c_(s)c_(s)a_(s)c_(s)t_(s)g_(s)

-3′ (SEQ ID NO: 597) 5′-AM-C6 ca

_(s)a_(s)c_(s)c_(s)a_(s)c_(s)t_(s)g_(s)

-   -   wherein uppercase letters denote beta-D-oxy-LNA units; lowercase        letters denote DNA units; the subscript “s” denotes a        phosphorothioate linkage; superscript m denotes a DNA or        beta-D-oxy-LNA unit containing a 5-methylcytosine base; AM-C6 is        an amino-C6 linker; wherein the 5′ terminal group “AM-C6 c a” is        optional.

-   79. The oligomer as defined in any of paragraphs 67 to 78 for use in    a medical treatment.

-   80. The oligomer as defined in any of paragraphs 67 to 79 for use in    the treatment of a viral disorder.

-   81. The oligomer as defined in paragraph 79 or paragraph 80 wherein    said oligomer is as defined in any one of paragraphs 1 to 53.

-   82. The oligomer as defined in paragraph 79 or paragraph 81 wherein    said disorder is as defined in any one of paragraphs 1 to 53.

-   83. A composition suitable for the treatment of a viral disorder,    wherein said composition comprises an oligomer and at least one    additional different oligonucleotide; wherein said oligomer    comprises at least one first oligomer region capable of modulating a    target sequence of Hepatitis B Virus (HBV), preferably HBx or HBsAg    of HBV, to treat said viral disorder.

-   84. A composition according to paragraph 83 wherein said oligomer is    an oligomer as defined in any one of paragraphs 67 to 82.

-   85. A composition according to paragraph 83 or paragraph 84 wherein    said at least one additional different oligonucleotide is an    additional different oligonucleotide as defined in any one of    paragraphs 54 to 61.

-   86. A method for treating a viral disorder, said method comprising    administering to a subject in need of treatment an effective amount    of an oligomer conjugate according to paragraph 62 or paragraph 63    or an oligomer conjugate as defined in any one of paragraphs 1 to    52.

-   87. A method for treating a viral disorder, said method comprising    administering to a subject in need of treatment an effective amount    of a composition according to any one of paragraphs 64 to 66 or a    composition as defined in any one of paragraphs 54 to 61 or a    composition as defined in any one of paragraphs 83 to 85.

-   88. A method for treating a viral disorder, said method comprising    administering to a subject in need of treatment an effective amount    of an oligomer according to any one of paragraphs 67 to 82.

-   89. A pharmaceutical composition comprising an oligomer conjugate    according to paragraph 62 or paragraph 63 or an oligomer conjugate    as defined in any one of paragraphs 1 to 53; and one or more    pharmaceutically acceptable diluents, carriers, salts or adjuvants.

-   90. A pharmaceutical composition comprising a composition according    to any one of paragraphs 64 to 66 or a composition as defined in any    one of paragraphs 54 to 61 or a composition as defined in any one of    paragraphs 83 to 85; and one or more pharmaceutically acceptable    diluents, carriers, salts or adjuvants.

-   91. A pharmaceutical composition comprising an oligomer according to    any one of paragraphs 67 to 82; and one or more pharmaceutically    acceptable diluents, carriers, salts or adjuvants.    -   A pharmaceutical system comprising a pharmaceutical composition        according to any one of paragraphs 89 to 91 and an additional        pharmaceutical entity.

-   92. A method of manufacturing an oligomer conjugate according to any    one of paragraphs 1 to 52, comprising conjugating one or more    oligomers as defined in any one of paragraphs 1 to 53 with a carrier    component as defined in any one of paragraphs 1 to 53.

-   93. A method of manufacturing a composition according to any one of    paragraphs 64 to 66, comprising admixing an oligomer conjugate as    defined in any one of paragraphs 1 to 53 with a pharmaceutically    acceptable diluent, carrier, salt or adjuvant.

-   94. A method of manufacturing a composition according to paragraph    91, comprising admixing an oligomer as defined in any one of    paragraphs 67 to 82 with a pharmaceutically acceptable diluent,    carrier, salt or adjuvant.

-   95. The invention according to any one of the preceding paragraphs    wherein the oligomer or oligomer component of the oligomer conjugate    comprises any one of the motifs: 3-10-3, 3-10-2, 3-9-3, 3-9-2,    3-8-3, 3-8-2 wherein the first number is the number of modified    nucleotides in the wing region, preferably at least one being an LNA    unit, preferably all being an LNA unit, the second number is the    number of nucleotides in the gap region, and the third number is the    number of modified nucleotides in the wing region, preferably at    least one being an LNA unit, preferably all being an LNA unit.

-   96. An oligomer conjugate substantially as described herein and with    reference to the Examples.

-   97. A composition substantially as described herein and with    reference to the Examples.

-   98. An oligomer substantially as described herein and with reference    to the Examples.

-   99. A method substantially as described herein and with reference to    the Examples.

Particular Embodiments

The present invention relates to an oligomer conjugate for use in thetreatment of a viral disorder. The oligomer conjugate comprises: a) anoligomer capable of modulating a target sequence in HBx or HBsAg ofHepatitis B Virus (HBV) to treat said viral disorder; and b) a carriercomponent conjugated to said oligomer. Preferably the carrier componentis for delivering said first oligomer to the liver.

Preferred aspects for certain embodiments of the present invention arenow provided.

In one aspect, the present invention provides an oligomer conjugate foruse in the treatment of a viral disorder, wherein said oligomerconjugate comprises:

-   -   a) at least one first oligomer region capable of modulating a        target sequence of Hepatitis B Virus (HBV), preferably HBx or        HBsAg of HBV, to treat said viral disorder; and    -   b) a carrier component;        wherein said first oligomer region is 8-16 nucleotides in        length;        wherein said first oligomer region is a gapmer, preferably        wherein said first oligomer region comprises a        2′-deoxyribonucleotide gap region flanked on each side by a        wing, preferably wherein each wing independently comprises one        or more LNA units;        wherein said carrier component is a carbohydrate conjugate        moiety, preferably said carrier component is selected from the        group consisting of galactose, galactosamine,        N-formyl-galactosamine, N-acetylgalactosamine (GalNAc),        N-propionyl-galactosamine, N-n-butanoyl-galactosamine,        N-isobutanoylgalactose-amine or a cluster of any one or more        thereof; preferably said carrier component comprises GalNAc or a        GalNAc cluster; preferably said carrier component is GalNAc2;        wherein said target sequence comprises at least part of a gene        or a mRNA encoding HBx or HBsAg or a naturally-occurring variant        thereof.

In one aspect, the present invention provides an oligomer conjugatesuitable for the treatment of a viral disorder, wherein said oligomerconjugate comprises:

-   -   a) at least one first oligomer region capable of modulating a        target sequence of Hepatitis B Virus (HBV), preferably HBx or        HBsAg of HBV, to treat said viral disorder; and    -   b) a carrier component        wherein said first oligomer region is 8-16 nucleotides in        length;        wherein said first oligomer region is a gapmer, preferably        wherein said first oligomer region comprises a        2′-deoxyribonucleotide gap region flanked on each side by a        wing, preferably wherein each wing independently comprises one        or more LNA units;        wherein said carrier component is a carbohydrate conjugate        moiety, preferably said carrier component is selected from the        group consisting of galactose, galactosamine,        N-formyl-galactosamine, N-acetylgalactosamine (GalNAc),        N-propionyl-galactosamine, N-n-butanoyl-galactosamine,        N-isobutanoylgalactose-amine or a cluster of any one or more        thereof; preferably said carrier component comprises GalNAc or a        GalNAc cluster; preferably said carrier component is GalNAc2;        wherein said target sequence comprises at least part of a gene        or a mRNA encoding HBx or HBsAg or a naturally-occurring variant        thereof.

In one aspect, the present invention provides a composition suitable forthe treatment of a viral disorder, wherein said composition comprises anoligomer conjugate and at least one additional differentoligonucleotide; wherein said oligomer conjugate comprises:

-   -   a) at least one first oligomer region capable of modulating a        target sequence of Hepatitis B Virus (HBV), preferably HBx or        HBsAg of HBV, to treat said viral disorder; and    -   b) a carrier component        wherein said first oligomer region is 8-16 nucleotides in        length;        wherein said first oligomer region is a gapmer, preferably        wherein said first oligomer region comprises a        2′-deoxyribonucleotide gap region flanked on each side by a        wing, preferably wherein each wing independently comprises one        or more LNA units;        wherein said carrier component is a carbohydrate conjugate        moiety, preferably said carrier component is selected from the        group consisting of galactose, galactosamine,        N-formyl-galactosamine, N-acetylgalactosamine (GalNAc),        N-propionyl-galactosamine, N-n-butanoyl-galactosamine,        N-isobutanoylgalactose-amine or a cluster of any one or more        thereof; preferably said carrier component comprises GalNAc or a        GalNAc cluster; preferably said carrier component is GalNAc2;        wherein said target sequence comprises at least part of a gene        or a mRNA encoding HBx or HBsAg or a naturally-occurring variant        thereof.

In one aspect, the present invention provides an oligomer conjugate foruse in the treatment of a viral disorder, wherein said oligomerconjugate comprises:

-   -   a) at least one first oligomer region capable of modulating a        target sequence of Hepatitis B Virus (HBV), preferably HBx or        HBsAg of HBV, to treat said viral disorder; and    -   b) a carrier component;        wherein said first oligomer region is 8-16 nucleotides in        length;        wherein said first oligomer region is a gapmer, preferably        wherein said first oligomer region comprises a        2′-deoxyribonucleotide gap region flanked on each side by a        wing, preferably wherein each wing independently comprises one        or more LNA units;        wherein said carrier component is a carbohydrate conjugate        moiety, preferably said carrier component is selected from the        group consisting of galactose, galactosamine,        N-formyl-galactosamine, N-acetylgalactosamine (GalNAc),        N-propionyl-galactosamine, N-n-butanoyl-galactosamine,        N-isobutanoylgalactose-amine or a cluster of any one or more        thereof; preferably said carrier component comprises GalNAc or a        GalNAc cluster; preferably said carrier component is GalNAc2;        wherein said target sequence comprises at least part of a gene        or a mRNA encoding HBx or HBsAg or a naturally-occurring variant        thereof;        wherein said first oligomer region is based on a core motif        selected from the group consisting of any one or more of:

(SEQ ID NO: 13) GCGTAAAGAGAGG; (SEQ ID NO: 11) GCGTAAAGAGAGGT;(SEQ ID NO: 20) AGCGAAGTGCACACG; (SEQ ID NO: 26) AGGTGAAGCGAAGTG;(SEQ ID NO 18) AGCGAAGTGCACACGG; (SEQ ID NO: 7) CGAACCACTGAACA;(SEQ ID NO 4) GAACCACTGAACAAA; (SEQ ID NO 5) CGAACCACTGAACAAA;(SEQ ID NO 6) CGAACCACTGAACAA; (SEQ ID NO 8) CGAACCACTGAAC (SEQ ID NO 9)CCGCAGTATGGATCG (SEQ ID NO: 10) CGCAGTATGGATC; (SEQ ID NO 12)CGCGTAAAGAGAGGT; (SEQ ID NO 14) AGAAGGCACAGACGG; (SEQ ID NO 15)GAGAAGGCACAGACGG (SEQ ID NO 16) GAAGTGCACACGG; (SEQ ID NO 17)GCGAAGTGCACACGG; (SEQ ID NO 19) CGAAGTGCACACG; (SEQ ID NO 27)AGGTGAAGCGAAGT; and (SEQ ID NO: 852) TAGTAAACTGAGCCA.

In one aspect, the present invention provides an oligomer conjugatesuitable for the treatment of a viral disorder, wherein said oligomerconjugate comprises:

-   -   a) at least one first oligomer region capable of modulating a        target sequence of Hepatitis B Virus (HBV), preferably HBx or        HBsAg of HBV, to treat said viral disorder; and    -   b) a carrier component        wherein said first oligomer region is 8-16 nucleotides in        length;        wherein said first oligomer region is a gapmer, preferably        wherein said first oligomer region comprises a        2′-deoxyribonucleotide gap region flanked on each side by a        wing, preferably wherein each wing independently comprises one        or more LNA units;        wherein said carrier component is a carbohydrate conjugate        moiety, preferably said carrier component is selected from the        group consisting of galactose, galactosamine,        N-formyl-galactosamine, N-acetylgalactosamine (GalNAc),        N-propionyl-galactosamine, N-n-butanoyl-galactosamine,        N-isobutanoylgalactose-amine or a cluster of any one or more        thereof; preferably said carrier component comprises GalNAc or a        GalNAc cluster; preferably said carrier component is GalNAc2;        wherein said target sequence comprises at least part of a gene        or a mRNA encoding HBx or HBsAg or a naturally-occurring variant        thereof;        wherein said first oligomer region is based on a core motif        selected from the group consisting of any one or more of:

(SEQ ID NO: 13) GCGTAAAGAGAGG; (SEQ ID NO: 20) AGCGAAGTGCACACG;(SEQ ID NO: 11) GCGTAAAGAGAGGT; (SEQ ID NO: 26) AGGTGAAGCGAAGTG;(SEQ ID NO 18) AGCGAAGTGCACACGG; (SEQ ID NO: 7) CGAACCACTGAACA;(SEQ ID NO 4) GAACCACTGAACAAA; (SEQ ID NO 5) CGAACCACTGAACAAA;(SEQ ID NO 6) CGAACCACTGAACAA; (SEQ ID NO 8) CGAACCACTGAAC (SEQ ID NO 9)CCGCAGTATGGATCG (SEQ ID NO: 10) CGCAGTATGGATC; (SEQ ID NO 12)CGCGTAAAGAGAGGT; (SEQ ID NO 14) AGAAGGCACAGACGG; (SEQ ID NO 15)GAGAAGGCACAGACGG (SEQ ID NO 16) GAAGTGCACACGG; (SEQ ID NO 17)GCGAAGTGCACACGG; (SEQ ID NO 19) CGAAGTGCACACG; (SEQ ID NO 27)AGGTGAAGCGAAGT; and (SEQ ID NO: 852 TAGTAAACTGAGCCA.

In one aspect, the present invention provides a composition suitable forthe treatment of a viral disorder, wherein said composition comprises anoligomer conjugate and at least one additional differentoligonucleotide; wherein said oligomer conjugate comprises:

-   -   a) at least one first oligomer region capable of modulating a        target sequence of Hepatitis B Virus (HBV), preferably HBx or        HBsAg of HBV, to treat said viral disorder; and    -   b) a carrier component        wherein said first oligomer region is 8-16 nucleotides in        length;        wherein said first oligomer region is a gapmer, preferably        wherein said first oligomer region comprises a        2′-deoxyribonucleotide gap region flanked on each side by a        wing, preferably wherein each wing independently comprises one        or more LNA units;        wherein said carrier component is a carbohydrate conjugate        moiety, preferably said carrier component is selected from the        group consisting of galactose, galactosamine,        N-formyl-galactosamine, N-acetylgalactosamine (GalNAc),        N-propionyl-galactosamine, N-n-butanoyl-galactosamine,        N-isobutanoylgalactose-amine or a cluster of any one or more        thereof; preferably said carrier component comprises GalNAc or a        GalNAc cluster; preferably said carrier component is GalNAc2;        wherein said target sequence comprises at least part of a gene        or a mRNA encoding HBx or HBsAg or a naturally-occurring variant        thereof;        wherein said first oligomer region is based on a core motif        selected from the group consisting of any one or more of:

(SEQ ID NO: 13) GCGTAAAGAGAGG; (SEQ ID NO: 11) GCGTAAAGAGAGGT;(SEQ ID NO: 20) AGCGAAGTGCACACG; (SEQ ID NO: 26) AGGTGAAGCGAAGTG;(SEQ ID NO 18) AGCGAAGTGCACACGG; (SEQ ID NO: 7) CGAACCACTGAACA;(SEQ ID NO 4) GAACCACTGAACAAA; (SEQ ID NO 5) CGAACCACTGAACAAA;(SEQ ID NO 6) CGAACCACTGAACAA; (SEQ ID NO 8) CGAACCACTGAAC (SEQ ID NO 9)CCGCAGTATGGATCG (SEQ ID NO: 10) CGCAGTATGGATC; (SEQ ID NO 12)CGCGTAAAGAGAGGT; (SEQ ID NO 14) AGAAGGCACAGACGG; (SEQ ID NO 15)GAGAAGGCACAGACGG (SEQ ID NO 16) GAAGTGCACACGG; (SEQ ID NO 17)GCGAAGTGCACACGG; (SEQ ID NO 19) CGAAGTGCACACG; (SEQ ID NO 27)AGGTGAAGCGAAGT; and (SEQ ID NO: 852 TAGTAAACTGAGCCA.

In one aspect, the present invention provides an oligomer conjugate foruse in the treatment of a viral disorder, wherein said oligomerconjugate comprises:

-   -   a) at least one first oligomer region capable of modulating a        target sequence of Hepatitis B Virus (HBV), preferably HBx or        HBsAg of HBV, to treat said viral disorder; and    -   b) a carrier component;        wherein said first oligomer region is 12-16 nucleotides in        length;        wherein said first oligomer region is a gapmer, preferably        wherein said first oligomer region comprises a        2′-deoxyribonucleotide gap region flanked on each side by a        wing, preferably wherein each wing independently comprises one        or more LNA units;        wherein said carrier component is a carbohydrate conjugate        moiety, preferably said carrier component is selected from the        group consisting of galactose, galactosamine,        N-formyl-galactosamine, N-acetylgalactosamine (GalNAc),        N-propionyl-galactosamine, N-n-butanoyl-galactosamine,        N-isobutanoylgalactose-amine or a cluster of any one or more        thereof; preferably said carrier component comprises GalNAc or a        GalNAc cluster; preferably said carrier component is GalNAc2;        wherein said target sequence comprises at least part of a gene        or a mRNA encoding HBx or HBsAg or a naturally-occurring variant        thereof;        wherein the oligomer component of the oligomer conjugate        comprises any one of the motifs: 3-10-3, 3-10-2, 3-9-3, 3-9-2,        3-8-3, 3-8-2 wherein the first number is the number of LNA units        in the wing region, the second number is the number of        nucleotides in the gap region, and the third number is the        number of LNA units in the wing region.

In one aspect, the present invention provides an oligomer conjugatesuitable for the treatment of a viral disorder, wherein said oligomerconjugate comprises:

-   -   a) at least one first oligomer region capable of modulating a        target sequence of Hepatitis B Virus (HBV), preferably HBx or        HBsAg of HBV, to treat said viral disorder; and    -   b) a carrier component        wherein said first oligomer region is 12-16 nucleotides in        length;        wherein said first oligomer region is a gapmer, preferably        wherein said first oligomer region comprises a        2′-deoxyribonucleotide gap region flanked on each side by a        wing, preferably wherein each wing independently comprises one        or more LNA units;        wherein said carrier component is a carbohydrate conjugate        moiety, preferably said carrier component is selected from the        group consisting of galactose, galactosamine,        N-formyl-galactosamine, N-acetylgalactosamine (GalNAc),        N-propionyl-galactosamine, N-n-butanoyl-galactosamine,        N-isobutanoylgalactose-amine or a cluster of any one or more        thereof; preferably said carrier component comprises GalNAc or a        GalNAc cluster; preferably said carrier component is GalNAc2;        wherein said target sequence comprises at least part of a gene        or a mRNA encoding HBx or HBsAg or a naturally-occurring variant        thereof;        wherein the oligomer component of the oligomer conjugate        comprises any one of the motifs: 3-10-3, 3-10-2, 3-9-3, 3-9-2,        3-8-3, 3-8-2 wherein the first number is the number of LNA units        in the wing region, the second number is the number of        nucleotides in the gap region, and the third number is the        number of LNA units in the wing region.

In one aspect, the present invention provides a composition suitable forthe treatment of a viral disorder, wherein said composition comprises anoligomer conjugate and at least one additional differentoligonucleotide; wherein said oligomer conjugate comprises:

-   -   a) at least one first oligomer region capable of modulating a        target sequence of Hepatitis B Virus (HBV), preferably HBx or        HBsAg of HBV, to treat said viral disorder; and    -   b) a carrier component        wherein said first oligomer region is 12-16 nucleotides in        length;        wherein said first oligomer region is a gapmer, preferably        wherein said first oligomer region comprises a        2′-deoxyribonucleotide gap region flanked on each side by a        wing, preferably wherein each wing independently comprises one        or more LNA units;        wherein said carrier component is a carbohydrate conjugate        moiety, preferably said carrier component is selected from the        group consisting of galactose, galactosamine,        N-formyl-galactosamine, N-acetylgalactosamine (GalNAc),        N-propionyl-galactosamine, N-n-butanoyl-galactosamine,        N-isobutanoylgalactose-amine or a cluster of any one or more        thereof; preferably said carrier component comprises GalNAc or a        GalNAc cluster; preferably said carrier component is GalNAc2;        wherein said target sequence comprises at least part of a gene        or a mRNA encoding HBx or HBsAg or a naturally-occurring variant        thereof;        wherein the oligomer component of the oligomer conjugate        comprises any one of the motifs: 3-10-3, 3-10-2, 3-9-3, 3-9-2,        3-8-3, 3-8-2 wherein the first number is the number of LNA units        in the wing region, the second number is the number of        nucleotides in the gap region, and the third number is the        number of LNA units in the wing region.

In one aspect, the present invention provides an oligomer conjugate foruse in the treatment of a viral disorder, wherein said oligomerconjugate comprises:

-   -   a) at least one first oligomer region capable of modulating a        target sequence of Hepatitis B Virus (HBV), preferably HBx or        HBsAg of HBV, to treat said viral disorder; and    -   b) a carrier component;        wherein said first oligomer region is 12-16 nucleotides in        length;        wherein said first oligomer region is a gapmer, preferably        wherein said first oligomer region comprises a        2′-deoxyribonucleotide gap region flanked on each side by a        wing, preferably wherein each wing independently comprises one        or more LNA units;        wherein said carrier component is a carbohydrate conjugate        moiety, preferably said carrier component is selected from the        group consisting of galactose, galactosamine,        N-formyl-galactosamine, N-acetylgalactosamine (GalNAc),        N-propionyl-galactosamine, N-n-butanoyl-galactosamine,        N-isobutanoylgalactose-amine or a cluster of any one or more        thereof; preferably said carrier component comprises GalNAc or a        GalNAc cluster; preferably said carrier component is GalNAc2;        wherein said target sequence comprises at least part of a gene        or a mRNA encoding HBx or HBsAg or a naturally-occurring variant        thereof;        wherein said first oligomer region is based on a core motif        selected from the group consisting of any one or more of:

(SEQ ID NO: 13) GCGTAAAGAGAGG; (SEQ ID NO: 11) GCGTAAAGAGAGGT;(SEQ ID NO: 20) AGCGAAGTGCACACG; (SEQ ID NO: 26) AGGTGAAGCGAAGTG;(SEQ ID NO 18) AGCGAAGTGCACACGG; (SEQ ID NO: 7) CGAACCACTGAACA;(SEQ ID NO 4) GAACCACTGAACAAA; (SEQ ID NO 5) CGAACCACTGAACAAA;(SEQ ID NO 6) CGAACCACTGAACAA; (SEQ ID NO 8) CGAACCACTGAAC (SEQ ID NO 9)CCGCAGTATGGATCG (SEQ ID NO: 10) CGCAGTATGGATC; (SEQ ID NO 12)CGCGTAAAGAGAGGT; (SEQ ID NO 14) AGAAGGCACAGACGG; (SEQ ID NO 15)GAGAAGGCACAGACGG (SEQ ID NO 16) GAAGTGCACACGG; (SEQ ID NO 17)GCGAAGTGCACACGG; (SEQ ID NO 19) CGAAGTGCACACG; (SEQ ID NO 27)AGGTGAAGCGAAGT; and (SEQ ID NO: 852 TAGTAAACTGAGCCA;wherein the oligomer component of the oligomer conjugate comprises anyone of the motifs: 3-10-3, 3-10-2, 3-9-3, 3-9-2, 3-8-3, 3-8-2 whereinthe first number is the number of LNA units in the wing region, thesecond number is the number of nucleotides in the gap region, and thethird number is the number of LNA units in the wing region.

In one aspect, the present invention provides an oligomer conjugatesuitable for the treatment of a viral disorder, wherein said oligomerconjugate comprises:

-   -   a) at least one first oligomer region capable of modulating a        target sequence of Hepatitis B Virus (HBV), preferably HBx or        HBsAg of HBV, to treat said viral disorder; and    -   b) a carrier component        wherein said first oligomer region is 12-16 nucleotides in        length;        wherein said first oligomer region is a gapmer, preferably        wherein said first oligomer region comprises a        2′-deoxyribonucleotide gap region flanked on each side by a        wing, preferably wherein each wing independently comprises one        or more LNA units;        wherein said carrier component is a carbohydrate conjugate        moiety, preferably said carrier component is selected from the        group consisting of galactose, galactosamine,        N-formyl-galactosamine, N-acetylgalactosamine (GalNAc),        N-propionyl-galactosamine, N-n-butanoyl-galactosamine,        N-isobutanoylgalactose-amine or a cluster of any one or more        thereof; preferably said carrier component comprises GalNAc or a        GalNAc cluster; preferably said carrier component is GalNAc2;        wherein said target sequence comprises at least part of a gene        or a mRNA encoding HBx or HBsAg or a naturally-occurring variant        thereof;        wherein said first oligomer region is based on a core motif        selected from the group consisting of any one or more of:

(SEQ ID NO: 13) GCGTAAAGAGAGG; (SEQ ID NO: 11) GCGTAAAGAGAGGT;(SEQ ID NO: 20) AGCGAAGTGCACACG; (SEQ ID NO: 26) AGGTGAAGCGAAGTG;(SEQ ID NO 18) AGCGAAGTGCACACGG; (SEQ ID NO: 7) CGAACCACTGAACA;(SEQ ID NO 4) GAACCACTGAACAAA; (SEQ ID NO 5) CGAACCACTGAACAAA;(SEQ ID NO 6) CGAACCACTGAACAA; (SEQ ID NO 8) CGAACCACTGAAC (SEQ ID NO 9)CCGCAGTATGGATCG (SEQ ID NO: 10) CGCAGTATGGATC; (SEQ ID NO 12)CGCGTAAAGAGAGGT; (SEQ ID NO 14) AGAAGGCACAGACGG; (SEQ ID NO 15)GAGAAGGCACAGACGG (SEQ ID NO 16) GAAGTGCACACGG; (SEQ ID NO 17)GCGAAGTGCACACGG; (SEQ ID NO 19) CGAAGTGCACACG; (SEQ ID NO 27)AGGTGAAGCGAAGT; and (SEQ ID NO: 852) TAGTAAACTGAGCCA;wherein the oligomer component of the oligomer conjugate comprises anyone of the motifs: 3-10-3, 3-10-2, 3-9-3, 3-9-2, 3-8-3, 3-8-2 whereinthe first number is the number of LNA units in the wing region, thesecond number is the number of nucleotides in the gap region, and thethird number is the number of LNA units in the wing region.

In one aspect, the present invention provides a composition suitable forthe treatment of a viral disorder, wherein said composition comprises anoligomer conjugate and at least one additional differentoligonucleotide; wherein said oligomer conjugate comprises:

-   -   a) at least one first oligomer region capable of modulating a        target sequence of Hepatitis B Virus (HBV), preferably HBx or        HBsAg of HBV, to treat said viral disorder; and    -   b) a carrier component,        wherein said first oligomer region is 12-16 nucleotides in        length;        wherein said first oligomer region is a gapmer, preferably        wherein said first oligomer region comprises a        2′-deoxyribonucleotide gap region flanked on each side by a        wing, preferably wherein each wing independently comprises one        or more LNA units;        wherein said carrier component is a carbohydrate conjugate        moiety, preferably said carrier component is selected from the        group consisting of galactose, galactosamine,        N-formyl-galactosamine, N-acetylgalactosamine (GalNAc),        N-propionyl-galactosamine, N-n-butanoyl-galactosamine,        N-isobutanoylgalactose-amine or a cluster of any one or more        thereof; preferably said carrier component comprises GalNAc or a        GalNAc cluster; preferably said carrier component is GalNAc2;        wherein said target sequence comprises at least part of a gene        or a mRNA encoding HBx or HBsAg or a naturally-occurring variant        thereof;        wherein said first oligomer region is based on a core motif        selected from the group consisting of any one or more of:

(SEQ ID NO: 13) GCGTAAAGAGAGG; (SEQ ID NO: 11) GCGTAAAGAGAGGT;(SEQ ID NO: 20) AGCGAAGTGCACACG; (SEQ ID NO: 26) AGGTGAAGCGAAGTG;(SEQ ID NO 18) AGCGAAGTGCACACGG; (SEQ ID NO: 7) CGAACCACTGAACA;(SEQ ID NO 4) GAACCACTGAACAAA; (SEQ ID NO 5) CGAACCACTGAACAAA;(SEQ ID NO 6) CGAACCACTGAACAA; (SEQ ID NO 8) CGAACCACTGAAC (SEQ ID NO 9)CCGCAGTATGGATCG (SEQ ID NO: 10) CGCAGTATGGATC; (SEQ ID NO 12)CGCGTAAAGAGAGGT; (SEQ ID NO 14) AGAAGGCACAGACGG; (SEQ ID NO 15)GAGAAGGCACAGACGG (SEQ ID NO 16) GAAGTGCACACGG; (SEQ ID NO 17)GCGAAGTGCACACGG; (SEQ ID NO 19) CGAAGTGCACACG; (SEQ ID NO 27)AGGTGAAGCGAAGT; and (SEQ ID NO: 852 TAGTAAACTGAGCCA;wherein the oligomer component of the oligomer conjugate comprises anyone of the motifs: 3-10-3, 3-10-2, 3-9-3, 3-9-2, 3-8-3, 3-8-2 whereinthe first number is the number of LNA units in the wing region, thesecond number is the number of nucleotides in the gap region, and thethird number is the number of LNA units in the wing region.

In one aspect, the present invention provides an oligomer for thetreatment of a viral disorder, wherein said oligomer comprises at leastone first oligomer region capable of modulating a target sequence ofHepatitis B Virus (HBV), preferably HBx or HBsAg of HBV, to treat saidviral disorder; and wherein said first oligomer region is 12-16nucleotides in length;

wherein said first oligomer region is a gapmer, preferably wherein saidfirst oligomer region comprises a 2′-deoxyribonucleotide gap regionflanked on each side by a wing, preferably wherein each wingindependently comprises one or more LNA units;

wherein said first oligomer region is based on a core motif selectedfrom the group consisting of any one or more of:

(SEQ ID NO: 13) GCGTAAAGAGAGG; (SEQ ID NO: 11) GCGTAAAGAGAGGT;(SEQ ID NO: 20) AGCGAAGTGCACACG; (SEQ ID NO: 26) AGGTGAAGCGAAGTG;(SEQ ID NO 18) AGCGAAGTGCACACGG; (SEQ ID NO: 7) CGAACCACTGAACA;(SEQ ID NO 4) GAACCACTGAACAAA; (SEQ ID NO 5) CGAACCACTGAACAAA;(SEQ ID NO 6) CGAACCACTGAACAA; (SEQ ID NO 8) CGAACCACTGAAC (SEQ ID NO 9)CCGCAGTATGGATCG (SEQ ID NO: 10) CGCAGTATGGATC; (SEQ ID NO 12)CGCGTAAAGAGAGGT; (SEQ ID NO 14) AGAAGGCACAGACGG; (SEQ ID NO 15)GAGAAGGCACAGACGG (SEQ ID NO 16) GAAGTGCACACGG; (SEQ ID NO 17)GCGAAGTGCACACGG; (SEQ ID NO 19) CGAAGTGCACACG; (SEQ ID NO 27)AGGTGAAGCGAAGT; and (SEQ ID NO: 852 TAGTAAACTGAGCCA;wherein the oligomer comprises any one of the motifs: 3-10-3, 3-10-2,3-9-3, 3-9-2, 3-8-3, 3-8-2 wherein the first number is the number of LNAunits in the wing region, the second number is the number of nucleotidesin the gap region, and the third number is the number of LNA units inthe wing region.

In one aspect, the present invention provides an oligomer suitable forthe treatment of a viral disorder, wherein said oligomer comprises atleast one first oligomer region capable of modulating a target sequenceof Hepatitis B Virus (HBV), preferably HBx or HBsAg of HBV, to treatsaid viral disorder; and wherein said first oligomer region is 12-16nucleotides in length;

wherein said first oligomer region is a gapmer, preferably wherein saidfirst oligomer region comprises a 2′-deoxyribonucleotide gap regionflanked on each side by a wing, preferably wherein each wingindependently comprises one or more LNA units;

wherein said first oligomer region is based on a core motif selectedfrom the group consisting of any one or more of:

(SEQ ID NO: 13) GCGTAAAGAGAGG; (SEQ ID NO: 11) GCGTAAAGAGAGGT;(SEQ ID NO: 20) AGCGAAGTGCACACG; (SEQ ID NO: 26) AGGTGAAGCGAAGTG;(SEQ ID NO 18) AGCGAAGTGCACACGG; (SEQ ID NO: 7) CGAACCACTGAACA;(SEQ ID NO 4) GAACCACTGAACAAA; (SEQ ID NO 5) CGAACCACTGAACAAA;(SEQ ID NO 6) CGAACCACTGAACAA; (SEQ ID NO 8) CGAACCACTGAAC (SEQ ID NO 9)CCGCAGTATGGATCG (SEQ ID NO: 10) CGCAGTATGGATC; (SEQ ID NO 12)CGCGTAAAGAGAGGT; (SEQ ID NO 14) AGAAGGCACAGACGG; (SEQ ID NO 15)GAGAAGGCACAGACGG (SEQ ID NO 16) GAAGTGCACACGG; (SEQ ID NO 17)GCGAAGTGCACACGG; (SEQ ID NO 19) CGAAGTGCACACG; (SEQ ID NO 27)AGGTGAAGCGAAGT; and (SEQ ID NO: 852 TAGTAAACTGAGCCA;wherein the oligomer comprises any one of the motifs: 3-10-3, 3-10-2,3-9-3, 3-9-2, 3-8-3, 3-8-2 wherein the first number is the number of LNAunits in the wing region, the second number is the number of nucleotidesin the gap region, and the third number is the number of LNA units inthe wing region.

EXAMPLES

Materials and Methods

HBsAg and HBeAg Detection

Serum HBsAg and HBeAg level were determined in the serum of infectedAAV-HBV mouse using the HBsAg chemoluminescence immunoassay (CLIA) andthe HBeAg CLIA kit (Autobio diagnostics Co. Ltd., Zhengzhou, China, Cat.no. CL0310-2 and CL0312-2 respectively), according to the manufacturer'sprotocol. Briefly, 50 μl of serum was transferred to the respectiveantibody coated microtiter plate and 50 μl of enzyme conjugate reagentwas added. The plate was incubated for 60 min on a shaker at roomtemperature before all wells were washed six times with washing bufferusing an automatic washer. 25 μl of substrate A and then 25 μl ofsubstrate B was added to each well. The plate was incubated for 10 minat RT before luminescence was measured using an Envision luminescencereader. HBsAg is given in the unit IU/ml; where 1 ng HBsAg=1.14 IU.HBeAg is given in the unit NCU/ml serum.

HBV DNA Extraction and qPCR

Initially mice serum was diluted by a factor of 10 (1:10) with Phosphatebuffered saline (PBS). DNA was extracted using the MagNA Pure 96 (Roche)robot. 50 μl of the diluted serum was mixed in a processing cartridgewith 200 ul MagNA Pure 96 external lysis buffer (Roche, Cat. no.06374913001) and incubated for 10 minutes. DNA was then extracted usingthe “MagNA Pure 96 DNA and Viral Nucleic Acid Small Volume Kit” (Roche,Cat. no. 06543588001) and the “Viral NA Plasma SV external^(II)lysis2.0” protocol. DNA elution volume was 50 μl.

Quantification of extracted HBV DNA was performed using a Taqman qPCRmachine (ViiA7, life technologies). Each DNA sample was tested induplicate in the PCR. 5 μl of DNA sample was added to 15 μl of PCRmastermix containing 10 μl TaqMan Gene Expression Master Mix (AppliedBiosystems, Cat. no. 4369016), 0.5 μl PrimeTime XL qPCR Primer/Probe(IDT) and 4.5 μl distilled water in a 384 well plate and the PCR wasperformed using the following settings: UDG Incubation (2 min, 50° C.),Enzyme Activation (10 min, 95° C.) and PCR (40 cycles with 15 sec, 95°for Denaturing and 1 min, 60° C. for annealing and extension). DNA copynumbers were calculated from C_(t) values based on a HBV plasmid DNAstandard curve by the ViiA7 software.

Sequences for TaqMan Primers and Probes (IDT):

Forward core primer (F3_core): CTG TGC CTT GGG TGG CTT TReverse primer (R3_core): AAG GAA AGA AGT CAG AAG GCA AAATaqman probe (P3_core): 56-FAM/AGC TCC AAA /ZEN/TTC TTT ATA AGG GTC GATGTC CAT G/3IABkFQTissue Specific In Vitro Linker Cleavage Assay

FAM-labeled oligomers with the physiologically labile linker to betested (e.g. a DNA phosphodiester linker (PO linker)) are subjected toin vitro cleavage using homogenates of the relevant tissues (e.g. liveror kidney) and Serum.

The tissue and serum samples are collected from a suitable animal (e.g.mice, monkey, pig or rat) and homogenized in a homogenisation buffer(0.5% Igepal CA-630, 25 mM Tris pH 8.0, 100 mM NaCl, pH 8.0 (adjustedwith 1 N NaOH). The tissue homogenates and Serum are spiked witholigomer to concentrations of 200 μg/g tissue. The samples are incubatedfor 24 hours at 37° C. and thereafter the samples are extracted withphenol-chloroform. The solutions are subjected to AIE HPLC analyses on aDionex Ultimate 3000 using an Dionex DNApac p-100 column and a gradientranging from 10 mM-1 M sodium perchlorate at pH 7.5. The content ofcleaved and non-cleaved oligomer is determined against a standard usingboth a fluoresense detector at 615 nm and a uv detector at 260 nm.

S1 Nuclease Cleavage Assay

FAM-labelled oligomers with 51 nuclease susceptible linkers (e.g. a DNAphosphodiester linker (PO linker)) are subjected to in vitro cleavage in51 nuclease extract or Serum.

100 μM of the oligomer are subjected to in vitro cleavage by 51 nucleasein nuclease buffer (60 U pr. 100 μL) for 20 and 120 minutes. Theenzymatic activity is stopped by adding EDTA to the buffer solution. Thesolutions are subjected to AIE HPLC analyses on a Dionex Ultimate 3000using an Dionex DNApac p-100 column and a gradient ranging from 10 mM-1M sodium perchlorate at pH 7.5. The content of cleaved and non-cleavedoligomer is determined against a standard using both a fluoresensedetector at 615 nm and a uv detector at 260 nm.

Example 1 Construction of Conjugates

Oligonucleotides were synthesized on uridine universal supports orUnyLinker support from Kinovate using the phosphoramidite approach on aMerMade12 or an OligoMaker DNA/RNA synthesizerat 4 μmol scale. At theend of the synthesis, the oligonucleotides were cleaved from the solidsupport using aqueous ammonia for 1-2 hours at room temperature, andfurther deprotected for 16 hours at 65° C. The oligonucleotides werepurified by reverse phase HPLC (RP-HPLC) and characterized by UPLC, andthe molecular mass was further confirmed by ESI-MS. See below for moredetails.

Elongation of the Oligonucleotide

The coupling of β-cyanoethyl-phosphoramidites (DNA-A(Bz), DNA-G(ibu),DNA-C(Bz), DNA-T, LNA-5-methyl-C(Bz), LNA-A(Bz), LNA-G(dmf), LNA-T orAmino-C6 linker) was performed by using a solution of 0.1 M of the5′-O-DMT-protected phosphoramidite in acetonitrile and DCI(4,5-dicyanoimidazole) in acetonitrile (0.25 M) as activator. Thiolationfor introduction of phosphorthioate linkages was carried out usingxanthane hydride (0.01 M in acetonitrile/pyridine 9:1). Phosphordiesterlinkages were introduced using 0.02 M iodine in THF/Pyridine/water7:2:1. The rest of the reagents were the ones typically used foroligonucleotide synthesis. For post solid phase synthesis conjugation acommercially available C6 aminolinker phorphoramidite was used in thelast cycle of the solid phase synthesis and after deprotection andcleavage from the solid support the aminolinked deprotectedoligonucleotide was isolated. The conjugate was introduced viaactivation of the carboxylic acid and subsequent reaction with the amineon the 5′-end of the oligonucleotide using standard synthesis methods.

Purification by RP-HPLC:

The crude compound was purified by preparative RP-HPLC on a PhenomenexJupiter C18 10μ 150×10 mm column. 0.1 M ammonium acetate pH 8 andacetonitrile was used as buffers at a flow rate of 5 mL/min. Thecollected fractions were lyophilized to give the purified compoundtypically as a white solid.

Abbreviations

DCI: 4,5-Dicyanoimidazole

DCM: Dichloromethane

DMF: Dimethylformamide

DMT: 4,4′-Dimethoxytrityl

THF: Tetrahydrofurane

Bz: Benzoyl

Ibu: Isobutyryl

RP-HPLC: Reverse phase high performance liquid chromatography

Example 2 Testing In Vitro Efficacy Introduction

The HbsAg assay used in the following studies is a standard method. Itmeasures the amount of virus produced. It therefore measures a reductionin virus due to oligomers or oligomer conjugates targeting HBx or HBsAg.In addition, oligomers or oligomer conjugates that target the HBxtranscriptet will also target the HbsAg transcript (see also column 3and 4 in the results table).

Cell Lines

HepG2.2.15 cells were cultured in DMEM+Glutamax-I medium (Invitrogen,Carlsbad, Calif., USA), supplemented with 10% fetal bovine serum(Invitrogen) and G418 (Invitrogen) at a final concentration of 200 mg/Land maintained in 5% CO₂ at 37° C.

HBsAg Assay

HepG2.2.15 cells (a constitutively HBV-expressing cell line) were seededin duplicate into white, 96-well plates at 1.5×10⁴ cells/well. The cellswere treated with single concentrations of oligomers or with athree-fold serial dilution series of the compounds in DMSO. The finalDMSO concentration in all wells was 1% and DMSO was used as control.

The HBsAg chemiluminescence immunoassay (CLIA) kit (Autobio DiagnosticsCo., Zhengzhou, China) was used to measure the levels of secreted HBVantigens semi-quantitatively. For the detection 50 uL/well culturesupernatant was used and the procedure conducted as directed bymanufacturer's instructions. The cytotoxicity was measured usingCellTiter-Glo (Promega, Madison, Wis., USA, Cat #G7571). Using theE-WorkBook Suite (ID Business Solutions Ltd., Guildford, UK)dose-response curves were generated and the IC₅₀ and CC₅₀ valuesextrapolated. The IC₅₀ and CC₅₀ are defined as the compoundconcentration (or conditioned media log dilution) at which HBsAgsecretion (IC50) and cytotoxicity (CC50), respectively, are reduced by50% compared to the control. Data may be presented as the EC50 value foran oligomer, when testing at a range of concentrations, or as theabsolute level of HBsAg in the supernatant as a percent of the HBsAglevels in the no drug control samples, when testing at a singleconcentration.

Results from Single Concentration Treatments

A total of 290 oligomers without conjugate were screened in the in vitroefficacy assay using a single dose of 25 μM oligomer. HBV antigen(HBsAg) secretion was measured after 13 days. Table 5 below show theresults of the screening. The oligomers of SEQ ID NO 294 to SEQ ID NO318 all reduced the HBsAg activity to less than 40% of the control. Theoligomer of SEQ ID NO 584 corresponds to the oligomer disclosed as SEQID NO 16 in U.S. Pat. No. 8,598,334.

An additional 213 oligomers were screened in the in vitro efficacy assayusing a single dose of 25 μM oligomer. The results are shown in FIG. 5 a.

TABLE 5 HBsAg activity of 25 μM oligomer as % of control. LNA oligomersequences (Upper case letters = beta- D-oxy LNA, C LNA is5-methyl C LNA, lower case letters = DNA, ^(m)c = 5- ActivityOligomers that Oligomers that methylcytosine DNA, (HBsAg levelsbind both bind HBsAg Oligomer all internucleoside in culture the HBsAgtranscripts start linkages are supernatant and the HBx (SEQ ID NO 3),position phosphorothiate as percent of transcript but not HBx U95551internucleoside DMSO treated standard SeqID (SEQ ID NO 2) transcript(SEQ ID NO 1) linkages) cells) dev SeqID 294 X  691 GAAccactgaacAAA 22.07  2.28 SeqID 295 X  691 CGAaccactgaacAAA  18.38  2.08 SeqID 296 X 692 CGAaccactgaaCAA  10.28  0.83 SeqID 297 X  693 CGAaccactgaACA  14.74 1.99 SeqID 298 X  694 CGAaccactgaAC  15.68  1.09 SeqID 299 x 1264CCGcagtatggaTCG  38.83  4.40 SeqID 300 x 1265 CGCagtatggaTC  26.86  2.15SeqID 301 x 1530 GCGtaaagagaGGT  34.38  1.52 SeqID 302 x 1530CGCgtaaagagaGGT  38.30  6.35 SeqID 303 x 1531 GCGtaaagagaGG  37.42  3.29SeqID 304 x 1551 AGAaggcacagaCGG  25.28  1.62 SeqID 305 x 1551GAGaaggcacagaCGG  25.02  1.51 SeqID 306 x 1577 GAAgtgcaca^(m)cGG  14.81 3.11 SeqID 307 x 1577 GCGaagtgcacaCGG  21.88  2.55 SeqID 308 x 1577AGCgaagtgcacaCGG  16.33  2.31 SeqID 309 x 1578 CGAagtgcacaCG  25.64 2.12 SeqID 310 x 1578 AGCgaagtgcacACG  23.45  1.99 SeqID 311 x 1578AAG^(m)cgaagtgcacACG  31.48  2.85 SeqID 312 x 1580 GAAg^(m)cgaagtgcACA 35.14  0.93 SeqID 313 x 1582 GGTgaag^(m)cgaagtGCA  38.27  2.92SeqID 314 x 1583 GGTgaag^(m)cgaagTGC  30.58  4.73 SeqID 315 x 1583AGGtgaag^(m)cgaagTGC  15.21  1.90 SeqID 316 x 1584 AGGtgaag^(m)cgaaGTG 13.27  0.84 SeqID 317 x 1585 AGGtgaag^(m)cgaAGT  13.29  0.75 SeqID 318x 1588 CAGaggtgaagCGA  32.61  2.06 SeqID 319 X  201 AAAaccc^(m)cgccTGT 72.50  5.31 SeqID 320 X  202 AAAaccc^(m)cgccTG  69.30  7.61 SeqID 321 X 245 ACGagtctagacTCT  99.91  3.04 SeqID 322 X  245 CACgagtctagacTCT 88.47  3.67 SeqID 323 X  246 ACGagtctagaCTC  96.60  5.49 SeqID 324 X 246 CACgagtctagaCTC  94.04  2.94 SeqID 325 X  246 CCA^(m)cgagtctagaCTC 75.51  2.45 SeqID 326 X  247 ACGagtctagaCT  75.87  2.71 SeqID 327 X 247 CACgagtctagACT  85.96  5.46 SeqID 328 X  247 CCA^(m)cgagtctagACT 65.26  8.04 SeqID 329 X  247 ACCa^(m)cgagtctagACT  73.80  4.94SeqID 330 X  248 ACgagtctagAC  92.49  5.72 SeqID 331 X  248CACgagtctagAC  83.99  4.30 SeqID 332 X  248 CCA^(m)cgagtctaGAC  72.31 5.85 SeqID 333 X  248 ACCa^(m)cgagtctaGAC  69.87  4.09 SeqID 334 X  248CACca^(m)cgagtctaGAC  74.68  5.33 SeqID 335 X  249 ACCa^(m)cgagtctAGA 78.52  3.55 SeqID 336 X  249 CACca^(m)cgagtctAGA  78.82  0.55 SeqID 337X  249 CCAcca^(m)cgagtctAGA  70.68 10.64 SeqID 338 X  250CCAcca^(m)cgagtcTAG  72.06  4.76 SeqID 339 X  250 TCCacca^(m)cgagtcTAG 96.44 19.49 SeqID 340 X  251 CCAcca^(m)cgagtCTA  62.36  3.29 SeqID 341X  251 TCCacca^(m)cgagtCTA  74.38 12.81 SeqID 342 X  251GTCcacca^(m)cgagtCTA  66.96  8.93 SeqID 343 X  252 TCCacca^(m)cgagTGT 88.72  6.91 SeqID 344 X  252 GTCcacca^(m)cgagTCT  81.54  3.34 SeqID 345X  252 AGTccacca^(m)cgagTCT  73.05  5.43 SeqID 346 X  253GTCcacca^(m)cgaGTC  86.41  4.93 SeqID 347 X  253 AGTccacca^(m)cgaGTC 68.79  2.19 SeqID 348 X  253 AAGtccacca^(m)cgaGTC  55.97  4.51SeqID 349 X  254 AGTccacca^(m)cgAGT  74.01  6.04 SeqID 350 X  254AAGtccacca^(m)cgAGT  64.64  2.31 SeqID 351 X  254 GAAgtccacca^(m)cgAGT 71.86  2.47 SeqID 352 X  255 AAGtccacca^(m)cgAG  74.30  9.08 SeqID 353X  255 GAAgtccacca^(m)cgAG  72.50  1.71 SeqID 354 X  255AGAagtccacca^(m)cgAG  70.48  1.43 SeqID 355 X  256 AGAagtccaccaCGA 60.35  2.15 SeqID 356 X  256 GAGaagtccaccaCGA  50.28  3.88 SeqID 357 X 257 GAGaagtccaccACG  67.82  4.14 SeqID 358 X  257 AGAgaagtccaccACG 67.76  2.44 SeqID 359 X  258 GAGagaagtccacCAC  62.90  3.23 SeqID 360 X 259 GAGagaagtccaCCA  74.92  3.15 SeqID 361 X  259 TGAgagaagtccaCCA 73.90  3.92 SeqID 362 X  260 GAGagaagtccACC  87.37  7.72 SeqID 363 X 260 TGAgagaagtccACC  65.36  4.30 SeqID 364 X  261 TGAgagaagtcCAC 109.4511.99 SeqID 365 X  384 AAAa^(m)cgc^(m)cgcaGA  70.63 10.93 SeqID 366 X 384 TAAaa^(m)cgc^(m)cgcAGA  76.98 11.99 SeqID 367 X  384ATAaaa^(m)cgc^(m)cgcAGA  67.24  7.92 SeqID 368 X  384GATaaaa^(m)cgc^(m)cgcAGA  35.91  1.17 SeqID 369 X  385ATAaaa^(m)cgc^(m)cgCAG  80.52  2.79 SeqID 370 X  385GATaaaa^(m)cgc^(m)cgCAG  57.46  3.54 SeqID 371 X  385TGAtaaaa^(m)cgc^(m)cgCAG  62.20  3.88 SeqID 372 X  386ATAaaa^(m)cgc^(m)cgCA  82.52  1.41 SeqID 373 X  386GATaaaa^(m)cgc^(m)cGCA  58.35  1.00 SeqID 374 X  386TGAtaaaa^(m)cgc^(m)cGCA  46.17  4.23 SeqID 375 X  386ATGataaaa^(m)cgc^(m)cGCA  42.47  2.86 SeqID 376 X  387ATaaaa^(m)cgc^(m)cGC  86.96  6.36 SeqID 377 X  387 GATaaaa^(m)cgc^(m)cGC 61.13  4.90 SeqID 378 X  387 TGAtaaaa^(m)cgcCGC  55.76  4.38 SeqID 379X  387 ATGataaaa^(m)cgcCGC  35.38  4.12 SeqID 380 X  388GAtaaaa^(m)cgcCG  71.71  3.20 SeqID 381 X  388 TGAtaaaa^(m)cgcCG  56.48 9.26 SeqID 382 X  388 ATGataaaa^(m)cgCCG  64.46  2.93 SeqID 383 X  389TGataaaa^(m)cgCC  87.42 18.65 SeqID 384 X  389 ATGataaaa^(m)cgCC  53.02 0.62 SeqID 385 X  390 ATgataaaa^(m)cGC 103.50  4.41 SeqID 386 X  411TAGcagcaggaTG  47.43  1.27 SeqID 387 X  411 ATAgcagcaggATG  59.29  9.28SeqID 388 X  411 CATagcagcaggATG  52.52  5.05 SeqID 389 X  411GCAtagcagcaggATG  40.12  5.45 SeqID 390 X  412 GCAtagcagcagGAT  33.12 2.79 SeqID 391 X  412 GGCatagcagcagGAT  34.61  1.30 SeqID 392 X  414GAGgcatagcagcAGG  57.92  6.48 SeqID 393 X  415 TGAggcatagcagCAG  44.07 1.35 SeqID 394 X  416 TGAggcatagcaGCA  59.87  0.84 SeqID 395 X  416ATGaggcatagcaGCA  57.12  5.36 SeqID 396 X  417 TGAggcatagcAGC  60.89 4.54 SeqID 397 X  417 ATGaggcatagcAGC  53.18  3.00 SeqID 398 X  417GATgaggcatagcAGC  35.17  4.19 SeqID 399 X  418 GATgaggcatagCAG  51.50 4.39 SeqID 400 X  418 AGAtgaggcatagCAG  30.80  2.00 SeqID 401 X  419GATgaggcataGCA  56.86  2.93 SeqID 402 X  419 AGAtgaggcataGCA  26.45 3.15 SeqID 403 X  419 AAGatgaggcataGCA  42.05  1.52 SeqID 404 X  422AAGaagatgaggcATA  50.42  4.13 SeqID 405 X  423 AAGaagatgaggCAT  47.84 4.75 SeqID 406 X  601 TGGgatgggaatACA 127.91 11.95 SeqID 407 X  601ATGggatgggaatACA 104.41  6.06 SeqID 408 X  602 TGGgatgggaaTAC  97.8633.35 SeqID 409 X  602 ATGggatgggaaTAC 100.98 11.30 SeqID 410 X  602GATgggatgggaaTAC  80.86  4.66 SeqID 411 X  603 ATGggatgggaATA 101.5617.94 SeqID 412 X  603 GATgggatgggaATA  95.71 10.78 SeqID 413 X  604GATgggatgggAAT 136.89  7.88 SeqID 414 X  691 AACcactgaacAAA  56.01  3.86SeqID 415 X  695 CGaaccactgAA  67.83  3.12 SeqID 416 X  708GGGggaaagccCT  61.65 34.06 SeqID 417 X  708 TGGgggaaagcCCT 166.53  7.16SeqID 418 X 1142 GCAa^(m)cggggtaaAGG  99.66  2.38 SeqID 419 X 1143GCAa^(m)cggggtaAAG 128.31 18.17 SeqID 420 X 1144 GCAa^(m)cggggtaAA142.61  5.42 SeqID 421 X 1176 AGCaaacacttgGCA  81.61  3.37 SeqID 422 X1176 CAGcaaacacttgGCA  59.14  4.84 SeqID 423 X 1177 CAGcaaacacttGGC 47.64  0.61 SeqID 424 X 1177 TCAgcaaacacttGGC  28.43  2.09 SeqID 425 X1178 TCAgcaaacactTGG  53.37  9.79 SeqID 426 x 1264 GCAgtatggatCG  51.14 8.21 SeqID 427 x 1264 CGCagtatggaTCG  45.10 12.21 SeqID 428 x 1264TCCgcagtatggaTCG  40.99  6.50 SeqID 429 x 1265 CCGcagtatggATC 144.1811.10 SeqID 430 x 1265 TCCgcagtatggATC  74.40  9.23 SeqID 431 x 1266CGcagtatggAT  77.64  7.37 SeqID 432 x 1266 CCGcagtatggAT  86.57  3.84SeqID 433 x 1266 TCCgcagtatgGAT  86.85 16.10 SeqID 434 x 1267TCCgcagtatgGA  90.43  0.25 SeqID 435 x 1269 TTc^(m)cgcagtaTG  70.69 5.10 SeqID 436 x 1530 CGTaaagagagGT  65.75  2.97 SeqID 437 x 1530CCG^(m)cgtaaagagaGGT  62.74  2.15 SeqID 438 x 1531 CGtaaagagaGG  73.84 4.24 SeqID 439 x 1531 CGCgtaaagagAGG  93.28  8.65 SeqID 440 x 1531CCG^(m)cgtaaagagAGG  64.33  0.96 SeqID 441 x 1532 CGCgtaaagagAG  92.93 7.76 SeqID 442 x 1532 CCG^(m)cgtaaagaGAG  46.68  2.09 SeqID 443 x 1533CG^(m)cgtaaagaGA  96.70 11.51 SeqID 444 x 1533 CCG^(m)cgtaaagaGA  63.96 2.03 SeqID 445 x 1534 CCg^(m)cgtaaagAG  60.43  4.10 SeqID 446 x 1547GGCacaga^(m)cgggGAG 105.39  4.49 SeqID 447 x 1547 AGGcacaga^(m)cgggGAG 65.54  1.27 SeqID 448 x 1548 GGCacaga^(m)cggGGA 114.17  2.09 SeqID 449x 1548 AGGcacaga^(m)cggGGA  63.67  1.22 SeqID 450 x 1548AAGgcacaga^(m)cggGGA  52.62  1.97 SeqID 451 x 1549 AGGcacaga^(m)cgGGG117.50  7.77 SeqID 452 x 1549 AAGgcacaga^(m)cgGGG 105.77  3.94 SeqID 453x 1549 GAAggcacaga^(m)cgGGG 109.39  7.50 SeqID 454 x 1550AGAaggcacaga^(m)cGGG  60.92  7.17 SeqID 455 x 1552 GAGaaggcacagACG 43.34  2.63 SeqID 456 x 1577 CGAagtgcacaCGG  35.27  4.48 SeqID 457 x1578 GCGaagtgcacACG  49.25  1.43 SeqID 458 x 1579 GCGaagtgcacAC  65.82 7.11 SeqID 459 x 1579 AGCgaagtgcaCAC  64.47  2.76 SeqID 460 x 1579AAG^(m)cgaagtgcaCAC  42.82  5.42 SeqID 461 x 1579 GAAg^(m)cgaagtgcaCAC 37.15  4.92 SeqID 462 x 1580 AGCgaagtgcaCA  59.26  7.36 SeqID 463 x1580 AAG^(m)cgaagtgcACA  56.58  1.80 SeqID 464 x 1580TGAag^(m)cgaagtgcACA  58.81  5.72 SeqID 465 x 1581 AAG^(m)cgaagtgcAC 61.08 10.94 SeqID 466 x 1581 GAAg^(m)cgaagtgCAC  87.45 24.12 SeqID 467x 1581 TGAag^(m)cgaagtgCAC  70.27  2.31 SeqID 468 x 1581GTGaag^(m)cgaagtgCAC  91.50  6.40 SeqID 469 x 1582 AAg^(m)cgaagtgCA 75.07 12.27 SeqID 470 x 1582 GAAg^(m)cgaagtgCA  52.66  2.00 SeqID 471 x1582 TGAag^(m)cgaagtGCA  55.33  5.23 SeqID 472 x 1582GTGaag^(m)cgaagtGCA  45.56  1.33 SeqID 473 x 1583 TGAag^(m)cgaagtGC 47.33  2.27 SeqID 474 x 1583 GTGaag^(m)cgaagTGC  48.90  0.54 SeqID 475x 1584 GTGaag^(m)cgaagTG  44.41  3.30 SeqID 476 x 1584GGTgaag^(m)cgaaGTG  39.79  2.48 SeqID 477 x 1584 GAGgtgaag^(m)cgaaGTG 45.00  0.65 SeqID 478 x 1585 GTgaag^(m)cgaaGT  62.45  5.15 SeqID 479 x1585 GGTgaag^(m)cgaaGT  43.08  2.85 SeqID 480 x 1585 GAGgtgaag^(m)cgaAGT 55.57  2.43 SeqID 481 x 1585 AGAggtgaag^(m)cgaAGT  39.17  2.82SeqID 482 x 1586 AGAggtgaag^(m)cgAAG  41.25  0.88 SeqID 483 x 1586CAGaggtgaag^(m)cgAAG  53.21  3.02 SeqID 484 x 1587 AGAggtgaag^(m)cGAA 38.81  4.56 SeqID 485 x 1587 CAGaggtgaag^(m)cGAA  42.07  2.56 SeqID 486x 1587 GCAgaggtgaag^(m)cGAA  56.89  8.91 SeqID 487 x 1588GCAgaggtgaagCGA  53.71  4.36 SeqID 488 x 1588 TGCagaggtgaagCGA  70.80 5.16 SeqID 489 x 1589 TGCagaggtgaaGCG  92.30  7.94 SeqID 490 x 1589GTGcagaggtgaaGCG  70.51 10.18 SeqID 491 x 1590 CGTgcagaggtgaAGC 130.0513.54 SeqID 492 x 1591 CGTgcagaggtgAAG  91.47 26.28 SeqID 493 x 1591ACGtgcagaggtgAAG  90.23  8.21 SeqID 494 x 1592 CGTgcagaggtGAA  64.8522.74 SeqID 495 x 1592 ACGtgcagaggtGAA  58.06  4.58 SeqID 496 x 1593CGTgcagaggtGA  81.44 11.66 SeqID 497 x 1593 ACGtgcagaggTGA  50.58  6.38SeqID 498 x 1616 CGTtca^(m)cggtgGT  54.29  3.89 SeqID 499 x 1690CTCaaggt^(m)cggTC  68.75  3.36 SeqID 500 x 1691 CCTcaaggt^(m)cgGT 110.10 6.42 SeqID 501 x 1691 GCCtcaaggt^(m)cGGT  94.30  7.43 SeqID 502 x 1706ACAgtctttgaaGTA  90.33 13.01 SeqID 503 x 1783 TTTatgcctacAG  98.15  8.26SeqID 504 x 1784 AATttatgcctACA 115.05  4.58 SeqID 505 x 1785AATttatgcctAC 126.86  2.63 SeqID 506 x 1787 CCAatttatgcCT 152.55 29.03SeqID 507 x 1865 GCTtggaggcttGAA 103.91  5.71 SeqID 508 x 1865AGCttggaggcttGAA 133.58  0.46 SeqID 509 x 1866 GCTtggaggctTGA  79.26 8.08 SeqID 510 x 1866 AGCttggaggctTGA 122.38  3.30 SeqID 511 x 1866CAGcttggaggctTGA 132.43 18.26 SeqID 512 x 1867 GCTtggaggctTG  81.8310.69 SeqID 513 x 1867 AGCttggaggcTTG  98.04  9.53 SeqID 514 x 1867CAGcttggaggcTTG 113.24  9.04 SeqID 515 x 1867 ACAgcttggaggcTTG 145.1511.39 SeqID 516 x 1868 CACagcttggaggCTT 122.78  2.77 SeqID 517 x 1869CACagcttggagGCT 123.68 22.48 SeqID 518 x 1869 GCAcagcttggagGCT 145.75 6.22 SeqID 519 x 1870 GCAcagcttggaGGC 146.57 13.30 SeqID 520 x 1870GGCacagcttggaGGC 127.49  1.31 SeqID 521 x 1871 AGGcacagcttggAGG 126.4411.99 SeqID 522 x 1872 AGGcacagcttgGAG 113.11  1.53 SeqID 523 x 1872AAGgcacagcttgGAG  97.13  2.46 SeqID 524 x 1873 AAGgcacagcttGGA 102.44 3.67 SeqID 525 x 1873 CAAggcacagcttGGA 112.11  6.26 SeqID 526 x 1874AAGgcacagctTGG 111.03  4.15 SeqID 527 x 1874 CAAggcacagctTGG 108.76 6.19 SeqID 528 x 1874 CCAaggcacagctTGG 111.82 10.50 SeqID 529 x 1875CAAggcacagcTTG 111.24  3.42 SeqID 530 x 1875 CCAaggcacagcTTG 113.32 5.72 SeqID 531 x 1876 CCAaggcacagCTT  93.54  9.51 SeqID 532 X 2272TGCgaatccacAC 111.18  2.58 SeqID 533 X 2272 GTG^(m)cgaatccaCAC 116.70 3.36 SeqID 534 X 2370 GGAgttcttcttCTA 117.09  3.11 SeqID 535 X 2370GGGagttcttcttCTA 138.82  2.94 SeqID 536 X 2371 GGGagttcttctTCT 112.95 5.88 SeqID 537 X 2371 AGGgagttcttctTCT 115.73  2.39 SeqID 538 X 2372AGGgagttcttcTTC 115.60 13.69 SeqID 539 X 2372 GAGggagttcttcTTC 160.5212.84 SeqID 540 X 2373 AGGgagttcttCTT 187.99 14.17 SeqID 541 X 2373GAGggagttcttCTT 152.23  3.57 SeqID 542 X 2373 CGAgggagttcttCTT 125.2910.56 SeqID 543 X 2374 CGAgggagttctTCT 125.58 12.12 SeqID 544 X 2374GCGagggagttctTCT 127.13  7.13 SeqID 545 X 2375 GCGagggagttcTTC 140.39 2.94 SeqID 546 X 2375 GGCgagggagttcTTC 103.21 15.13 SeqID 547 X 2376GCGagggagttCTT 104.72 31.01 SeqID 548 X 2376 GGCgagggagttCTT 120.43 4.29 SeqID 549 X 2376 AGG^(m)cgagggagttCTT 126.12  8.49 SeqID 550 X2377 GCGagggagttCT  99.63 21.49 SeqID 551 X 2377 GGCgagggagtTCT 126.46 5.07 SeqID 552 X 2377 AGG^(m)cgagggagtTCT 124.41  7.46 SeqID 553 X 2377GAGg^(m)cgagggagtTCT 122.05  4.13 SeqID 554 X 2378 GGCgagggagtTC  93.7532.13 SeqID 555 X 2378 AGG^(m)cgagggagTTC  97.91 10.99 SeqID 556 X 2378GAGg^(m)cgagggagTTC 125.97 14.10 SeqID 557 X 2378 CGAgg^(m)cgagggagTTC114.85 11.32 SeqID 558 X 2379 AGG^(m)cgagggagTT 161.79 17.40 SeqID 559 X2379 GAGg^(m)cgagggaGTT 112.49 14.53 SeqID 560 X 2379CGAgg^(m)cgagggaGTT  99.73  6.03 SeqID 561 X 2379 GCGagg^(m)cgagggaGTT 26.82 16.61 SeqID 562 X 2380 GAGg^(m)cgagggaGT 126.11 10.60 SeqID 563 X2380 CGAgg^(m)cgagggAGT 113.92  2.48 SeqID 564 X 2380GCGagg^(m)cgagggAGT 114.21 22.83 SeqID 565 X 2380 TGCgagg^(m)cgagggAGT120.75  7.83 SeqID 566 X 2381 CGAgg^(m)cgagggAG  96.48  7.91 SeqID 567 X2381 GCGagg^(m)cgaggGAG 111.38 23.08 SeqID 568 X 2381TGCgagg^(m)cgaggGAG 150.61  9.91 SeqID 569 X 2381CTG^(m)cgagg^(m)cgaggGAG 115.39  3.45 SeqID 570 X 2382 CGagg^(m)cgaggGA 94.94  9.74 SeqID 571 X 2382 GCGagg^(m)cgaggGA 107.97 15.32 SeqID 572 X2382 TGCgagg^(m)cgagGGA  90.20 10.52 SeqID 573 X 2382CTG^(m)cgagg^(m)cgagGGA 117.46 22.79 SeqID 574 X 2382TCTg^(m)cgagg^(m)cgagGGA 119.40  1.22 SeqID 575 X 2383TCTg^(m)cgagg^(m)cgaGGG 139.98  6.20 SeqID 576 X 2383GTCtg^(m)cgagg^(m)cgaGGG  17.32  7.04 SeqID 577 X 2824 GTTcccaagaaTAT121.37  5.20 SeqID 578 X 2824 TGTtcccaagaaTAT 119.62  5.98 SeqID 579 X2825 GTTcccaagaaTA 118.81 12.04 SeqID 580 X 2825 TGTtcccaagaATA 119.1420.21 SeqID 581 X 2825 TTGttcccaagaATA 109.70  3.36 SeqID 582 X 2826TGTtcccaagaAT 110.02 17.04 SeqID 583 X 2826 TTGttcccaagAAT 132.22  4.26SeqID 584 X  414 GAGGcatagcagCAGG  79.64 10.52

TABLE 5a HBsAg activity of 25 μM oligomer as % of control.LNA oligomer sequnces (Upper case letters = beta-D-oxy LNA, C LNAis 5-methyl C LNA, lower case letters = DNA, Oligomer^(m)c = 5-methylcytosine DNA, start all internucleoside Activity (HBsAgposition linkages are  levels in culture U95551 phosphorothiatesupernatant as (SEQ ID internucleoside percent of DMSO standard SeqIDNO 1) linkages) treated cells) dev 585  691 GAAccactgaaCAAA  31.9  2.6586  691 GAACcactgaacAAA  35.1  1.3 587  691 CGaaccactgaaCAAA  45.5  2.4588  691 CGAAccactgaacAAA  18.3  2.2 589  691 CGAaccactgaaCAAA  36.2 9.1 590  691 CGAAccactgaacaAA  22.4  1.6 591  691 CGAAccactgaaCAAA 26.3  2.3 592  692 CGAAccactgaacAA  11.8  1.2 593  692 CGAAccactgaaCAA 21.9  8.5 594  692 CGAaccactgaACAA  18.3  0.6 595  693 CGaaccactgAACA 25.8  1.6 596  693 CGAAccactgaaCA   9.5  1.5 597  693 CGAaccactgAACA 23.6  2.0 598  693 CGAAccactgaACA  14.6  0.9 599  694 CGaaccactgAAC 42.1  3.8 600  694 CGAaccactgAAC  25.0  1.7 601 1264 CCgcagtatggATCG 98.7 16.4 602 1264 CCGCagtatggatCG  62.0  2.0 603 1264 CCGCagtatggaTCG 79.6 21.3 604 1264 CCGcagtatggATCG 113.3 12.3 605 1265 CGCAgtatggaTC 43.0  5.9 606 1265 CGcagtatggATC  97.7 22.6 607 1265 CGCagtatggATC 80.1 21.3 608 1265 CGcagtatgGATC 110.0 11.2 609 1530 GCGTaaagagagGT 65.2  6.6 610 1530 GCgtaaagagAGGT  43.4  6.1 611 1530 GCGtaaagagAGGT 59.9  5.8 612 1530 GCGTaaagagaGGT  52.7  1.9 613 1530 CGCGtaaagagagGT 96.7  9.7 614 1530 CG^(m)cgtaaagagAGGT  35.9  1.1 615 1530CGCGtaaagagaGGT  63.1  3.9 616 1530 CGCgtaaagagAGGT  65.0  4.6 617 1531GCgtaaagagAGG  24.6  1.5 618 1531 GCGtaaagagAGG  32.2  1.3 619 1531GCgtaaagaGAGG  54.7  4.7 620 1531 GCGTaaagagaGG  59.0  0.4 621 1551AGaaggcacagACGG  41.2  4.2 622 1551 AGAaggcacagACGG  45.4 10.9 623 1551AGAAggcacagaCGG  38.3  4.0 624 1551 GAGAaggcacagaCGG  35.0 11.1 625 1551GAGaaggcacagACGG  50.3 10.2 626 1551 GAGAaggcacagACGG  48.1  2.1 6271577 GAagtgcacaCGG  21.5  2.1 628 1577 GAAgtgcacaCGG  23.7  1.4 629 1577GAAGtgcacaCGG  41.2  1.3 630 1577 GAAgtgcacACGG  29.3  1.3 631 1577GCgaagtgcacaCGG  54.0 19.6 632 1577 GCGaagtgcaca^(m)cGG  49.7 10.7 6331577 GCGAagtgcaca^(m)cGG  30.3  5.2 634 1577 GCgaagtgcacACGG  46.9  7.3635 1577 AGCGaagtgcaca^(m)cGG  47.5 10.3 636 1577 AG^(m)cgaagtgcacACGG 38.1  4.2 637 1577 AG^(m)cgaagtgcacaCGG  83.2 37.3 638 1577AGCgaagtgcaca^(m)cGG  43.1 17.7 639 1578 CGaagtgcaCACG  58.3  6.9 6401578 CGAagtgcacACG  30.9  2.7 641 1578 CGaagtgcacACG  45.4  2.1 642 1578AGCgaagtgcaCACG 128.1  7.6 643 1578 AGCGaagtgcacACG  49.6  5.3 644 1578AGCGaagtgcacaCG  47.4  5.3 645 1578 AG^(m)cgaagtgcaCACG  59.1  3.5 6461578 AAg^(m)cgaagtgcaCACG  91.7 20.2 647 1578 AAGCgaagtgcacaCG  49.5 3.0 648 1578 AAG^(m)cgaagtgcaCACG  63.2  1.9 649 1578 AAGCgaagtgcacACG 41.9  2.1 650 1578 AAGCgaagtgcaCACG  64.7  4.5 651 1580GAag^(m)cgaagtgCACA 142.9 18.3 652 1580 GAAG^(m)cgaagtgcaCA  61.5  4.1653 1580 GAAg^(m)cgaagtgCACA 152.1 21.8 654 1580 GAAG^(m)cgaagtgCACA167.2 17.5 655 1582 GGtgaag^(m)cgaagtGCA 117.8 18.0 656 1582GGTgaag^(m)cgaagtgCA 108.0 10.4 657 1582 GGTGaag^(m)cgaagtgCA  93.5 24.7658 1582 GGtgaag^(m)cgaagTGCA 125.1 18.6 659 1583 GGtgaag^(m)cgaagTGC109.4 18.5 660 1583 GGTgaag^(m)cgaagtGC 104.1 14.0 661 1583GGTGaag^(m)cgaagtGC  84.4 23.1 662 1583 GGtgaag^(m)cgaaGTGC  60.1  9.7663 1583 AGgtgaag^(m)cgaagTGC  48.4  5.5 664 1583 AGGtgaag^(m)cgaagtGC 42.3  3.7 665 1583 AGGTgaag^(m)cgaagtGC  71.6 12.3 666 1583AGgtgaag^(m)cgaaGTGC  50.7 10.7 667 1584 AGGTgaag^(m)cgaagTG  47.7 44.6668 1584 AGgtgaag^(m)cgaAGTG  27.7  2.1 669 1584 AGGtgaag^(m)cgaAGTG 15.7  2.1 670 1584 AGGTgaag^(m)cgaaGTG  58.8 49.5 671 1585AGGTgaag^(m)cgaaGT 118.2 14.8 672 1585 AGgtgaag^(m)cgAAGT  31.5 38.6 6731585 AGGtgaag^(m)cgAAGT  25.8  4.4 674 1585 AGGTgaag^(m)cgaAGT  48.237.8 675 1588 CAGAggtgaag^(m)cGA  52.4  4.6 676 1588 CAgaggtgaaGCGA 67.4  0.1 677 1588 CAGaggtgaaGCGA  79.0  9.4 678  670 TAGtaaactgagCCA 31.3  2.7 679  670 TAgtaaactgaGCCA  93.0 12.1 680  670 TAGTaaactgagcCA 15.8  2.4 681  670 TAGtaaactgaGCCA  66.9  6.6 682  670 TAGTaaactgagCCA 26.6  4.9 683  670 CTAgtaaactgagCCA 101.6  6.7 684  670CTagtaaactgaGCCA 158.0 11.6 685  670 CTAGtaaactgagcCA 102.4 34.7 686 671 CTAgtaaactgaGCC  76.3 32.9 687  671 CTagtaaactgAGCC  53.0 13.8 688 671 CTAGtaaactgagCC  41.9  5.1 689  671 CTagtaaactgaGCC  31.8  2.2 690 671 CTAgtaaactgagCC 102.9 10.2 691  674 GCActagtaaacTGA  53.6  4.8 692 674 GCactagtaaaCTGA  74.4  1.5 693  674 GCACtagtaaactGA  81.9 10.6 694 674 GCActagtaaaCTGA  54.5  1.5 695  674 GCACtagtaaacTGA  74.9  4.5 696 674 GGCactagtaaacTGA  42.8 60.5 697  674 GGcactagtaaaCTGA  47.1 38.9698  674 GGCActagtaaactGA 147.7  7.1 699 1141 CAA^(m)cggggtaaaGGT 187.2 5.6 700 1141 CAa^(m)cggggtaaAGGT 176.2 18.0 701 1141 CAACggggtaaagGT187.9  3.8 702 1141 CAA^(m)cggggtaaAGGT 142.7 19.7 703 1141CAACggggtaaaGGT 144.8 31.8 704 1261 CAGtatggat^(m)cgGCA  59.6 19.9 7051261 CAgtatggat^(m)cGGCA  54.3  4.1 706 1261 CAgtatggat^(m)cgGCA  70.0 8.6 707 1261 CAGtatggat^(m)cggCA  60.3  8.0 708 1265TTC^(m)cgcagtatggATC 110.7  2.0 709 1265 TTc^(m)cgcagtatgGATC 105.5  4.2710 1265 TTCCgcagtatggaTC 104.1  6.7 711 1265 TTC^(m)cgcagtatgGATC 107.1 8.8 712 1265 TTCCgcagtatggATC 119.0  9.6 713 1266 TTC^(m)cgcagtatgGAT 99.8  6.8 714 1266 TTc^(m)cgcagtatGGAT  92.3  5.1 715 1266TTCCgcagtatggAT 104.7  3.6 716 1266 TTC^(m)cgcagtatGGAT 108.7  3.8 7171266 TTCCgcagtatgGAT 112.0  2.1 718 1266 GTTc^(m)cgcagtatgGAT  85.5  4.5719 1266 GTtc^(m)cgcagtatGGAT  80.0  6.7 720 1266 GTTC^(m)cgcagtatggAT127.9 16.9 721 1267 GTtc^(m)cgcagtaTGGA  67.2  5.0 722 1267GTTC^(m)cgcagtatgGA 150.8  5.9 723 1267 GTtc^(m)cgcagtatGGA  78.6  8.0724 1267 GTTc^(m)cgcagtatgGA  76.5  7.9 725 1267 AGTtc^(m)cgcagtatGGA 72.6  5.2 726 1267 AGTTc^(m)cgcagtatgGA  87.1  5.6 727 1267AGttc^(m)cgcagtatGGA  83.1  4.3 728 1267 AGTtc^(m)cgcagtatgGA  79.4  5.6729 1267 AGttc^(m)cgcagtatgGA  89.9  2.0 730 1268 AGTtc^(m)cgcagtaTGG 51.2  4.0 731 1268 AGttc^(m)cgcagtaTGG  63.6  0.5 732 1268AGTtc^(m)cgcagtatGG  65.9  1.6 733 1268 AGttc^(m)cgcagtatGG  80.9  2.5734 1268 GAgttc^(m)cgcagtaTGG  49.2  4.7 735 1268 GAGttc^(m)cgcagtatGG 60.1  6.1 736 1268 GAgttc^(m)cgcagtatGG  73.9  1.1 737 1269GAGTtc^(m)cgcagtaTG  58.6  6.6 738 1269 GAgttc^(m)cgcagtATG  57.1  8.1739 1269 GAGttc^(m)cgcagtaTG  49.8  6.8 740 1269 GAgttc^(m)cgcagtaTG 60.8  1.6 741 1269 GGAGttc^(m)cgcagtaTG 137.3  2.6 742 1269GGagttc^(m)cgcagtATG  90.5 22.8 743 1269 GGAgttc^(m)cgcagtaTG 117.5  2.4744 1269 GGagttc^(m)cgcagtaTG 124.7  6.4 745 1525 TAAagagaggtg^(m)cGCC 71.3 60.7 746 1525 TAaagagaggtgCGCC  73.9 59.0 747 1525TAAAgagaggtg^(m)cgCC  79.5 45.6 748 1525 TAAagagaggtgCGCC  93.6  4.6 7491525 TAAAgagaggtg^(m)cGCC  28.0 22.3 750 1526 TAAagagaggtgCGC  96.3 22.2751 1526 TAaagagaggtGCGC 101.9 73.2 752 1526 TAAagagaggtGCGC  44.3 72.6753 1526 TAAAgagaggtgCGC  64.1 50.8 754 1526 GTAaagagaggtgCGC  27.0 47.8755 1526 GTaaagagaggtGCGC  65.6 58.8 756 1527 GTAaagagaggtGCG  23.6 42.9757 1527 GTaaagagaggTGCG  80.5  2.4 758 1527 GTAaagagaggTGCG  81.8 61.8759 1527 GTAAagagaggtGCG  31.5 35.2 760 1527 CGtaaagagaggTGCG  91.5 62.5761 1527 CGTAaagagaggtgCG  70.5 60.0 762 1527 CGTaaagagaggTGCG  79.660.1 763 1527 CGTAaagagaggtGCG   4.5  4.1 764 1528 CGTaaagagaggTGC  37.055.1 765 1528 CGtaaagagagGTGC  89.4 17.7 766 1528 CGTAaagagaggtGC  93.317.1 767 1528 CGTaaagagagGTGC  63.4  4.2 768 1528 CGTAaagagaggTGC  34.151.7 769 1528 GCGtaaagagaggTGC  50.5 83.8 770 1528 GCgtaaagagagGTGC 43.6 73.2 771 1528 GCgtaaagagaggTGC  31.3 53.0 772 1528GCGtaaagagaggtGC  43.9 69.9 773 1529 GCGtaaagagagGTG  53.2 67.4 774 1529GCgtaaagagaGGTG   2.9  3.1 775 1529 GCGTaaagagaggTG  44.0 32.8 776 1529GCGtaaagagaGGTG   8.8  4.3 777 1529 GCGTaaagagagGTG  -0.6  0.6 778 1529CGCgtaaagagagGTG  43.2 40.7 779 1529 ^(m)cg^(m)cgtaaagagaGGTG   8.3  5.3780 1529 CGCGtaaagagaggTG  37.6 40.0 781 1529 CGCgtaaagagaGGTG  33.540.4 782 1529 CGCGtaaagagagGTG  33.5 54.0 783 1552 TGAgaaggcacagACG 95.2  5.5 784 1552 TGagaaggcacaGACG  54.4 48.4 785 1552TGAGaaggcacagaCG  67.2  6.3 786 1552 TGAgaaggcacaGACG  49.4 43.8 7871552 TGAGaaggcacagACG  56.1  9.5 788 1690 GCctcaaggt^(m)cgGTC  78.8 70.1789 1690 GCCtcaaggt^(m)cggTC  21.6 40.1 790 1690 GCctcaaggt^(m)cggTC 46.7 74.2 791 1778 ATgcctacagccTCC  51.8  1.4 792 1778 ATGcctacagcctCC 51.1  2.2 793 1778 ATgcctacagcctCC  59.4  7.0 794 1785 ACCAatttatgcCTAC145.3  4.8 795 1785 ACCaatttatgcCTAC 138.6 10.2 796 1785ACCAatttatgccTAC 137.4  0.4 797 1785 ACCaatttatgccTAC 131.3  9.2 7981785 ACcaatttatgcCTAC 126.0  8.7Results from Multiple Concentration Treatments

A selection of oligomers from Table 5 were tested using three-foldserial dilutions (25.000, 8.3333, 2.7778, 0.9259, 0.0343, 0.0114,0.0038, 0.0013 μM oligomer) in the in vitro efficacy assay to assess IC50 and CC50 values for the oligomers. HBV antigen (HBsAg) secretion wasmeasured after 13 days. Table 6 below show the results of the analysis.The oligomer of SEQ ID NO 585 corresponds to the oligomer disclosed asSEQ ID NO 16 in U.S. Pat. No. 8,598,334.

TABLE 6 Seq ID IC₅₀ (μM) CC₅₀ (μM) SeqID 294 2.07 >25 SeqID 295 2.05 >25SeqID 296 1.72 <0.0013 SeqID 297 0.45 >25 SeqID 298 0.44 >25 SeqID 3000.54 >25 SeqID 301 0.96 >25 SeqID 303 2.57 >25 SeqID 304 1.70 <0.0013SeqID 305 2.05 >25 SeqID 306 1.18 >25 SeqID 307 0.68 >25 SeqID 3082.62 >25 SeqID 309 2.15 >25 SeqID 310 2.04 >25 SeqID 311 9.75 >25 SeqID315 1.12 >25 SeqID 316 1.13 >25 SeqID 317 0.80 >25 SeqID 318 5.27 >25SeqID 368 >25 <0.0013 SeqID 386 12.32 >25 SeqID 389 23.43 >25 SeqID 3902.57 >25 SeqID 391 5.91 >25 SeqID 393 3.08 >25 SeqID 398 19.84 >25 SeqID400 3.07 >25 SeqID 402 2.00 >25 SeqID 424 2.43 >25 SeqID 427 0.61 >25SeqID 442 2.24 >25 SeqID 456 0.78 >25 SeqID 457 6.05 >25 SeqID 4733.91 >25 SeqID 474 4.67 >25 SeqID 475 6.15 >25 SeqID 476 3.82 >25 SeqID479 5.88 >25 SeqID 481 6.63 >25 SeqID 482 10.10 >25 SeqID 484 17.04 >25SeqID 485 4.34 >25 SeqID 584 >25 >25

Example 3 In Vivo AAV/HBV Mouse Model

Anti-HBV LNAs can be evaluated in AAV/HBV mouse model. In this model,mice infected with a recombinant adeno-associated virus (AAV) carryingthe HBV genome (AAV/HBV) maintains stable viremia and antigenimia formore than 30 weeks (Dan Yang, et al. 2014 Cellular & MolecularImmunology 11, 71-78).

Male C57BL/6 mice (4-6 weeks old), specific pathogen free, are purchasedfrom SLAC (Shanghai Laboratory Animal Center of Chinese Academy ofSciences) and housed in an animal care facility in individuallyventilated cages. Guidelines are followed for the care and use ofanimals as indicated by WuXi IACUC (Institutional Animal Care and UseCommittee, WUXI IACUC protocol number R20131126-Mouse). Mice are allowedto acclimate to the new environment for 3 days and are grouped accordingto the experimental design.

Recombinant AAV-HBV was diluted in PBS, 200 μL per injection. Thisrecombinant virus carries 1.3 copies of the HBV genome (genotype D,serotype ayw).

On day 0, all mice are injected through tail vein with 200 μL AAV-HBV.On days 6, 13 and 20 after AAV injection, all mice in aresubmandibularly bled (0.1 ml blood/mouse) for serum collection. On day22 post injection, mice with stable viremia are treated with vehicle oranti-HBV LNAs dosed intravenously at 5 mg/kg. The LNA oligomers can beunconjugated or GalNAc conjugated.

Mice are dosed biweekly for two weeks. On days 3, 7, 10 and 14 daysafter first LNA dosing, all mice are submandibularly bled (0.1 mlblood/mouse) for serum collection to monitor HBV surface antigen(HBsAg), HBV e antigen (HBeAg), and HBV genomic DNA in serum.

Example 4 In Vivo Study of Bi-Weekly Injections at Single Dose

The AAV/HBV Mouse Model as prepared in Example 3 was used in this study.Ten GalNAc conjugated Anti-HBV LNA oligomers were tested with saline ascontrol in C57BL/6 mice with stable viremia. Some of the oligomers werecompared with the standard of care nucleoside analog, Entecavir (ETV),administered as prescribed with 0.03 mg per kilo as a daily oral dosis.

Mice were dosed subcutaneously twice weekly for two weeks on days 0, 3,7 and 10 or day 0, 3, 6 and 9 with a dose of 2 mg/kg pr injection. HBVsurface antigen (HBsAg), HBV e antigen (HBeAg), and HBV genomic DNA inserum was measured at the indicated days using the methods described inthe “Materials and methods” section. The mice were followed for 23-24days.

The results are shown in the tables below.

TABLE 7A-C Serum level of HBsAg (log₁₀(IU/ml)) following twice-weeklydosages of 2 mg/kg Data are from three independent studies. A SalineSEQID806 SEQID807 SEQID815 SEQID800 SEQID802 ETV HBsAg HBsAg HBsAg HBsAgHBsAg HBsAg HBsAg St St St St St St St Day dev dev Dev Dev Dev dev dev 0 4.23 0.53 4.79 0.04 4.68 0.04 4.06 0.76 4.73 0.01 4.07 0.37 4.49 0.24 3 4.05 0.40 4.48 0.16 4.00 0.12 3.48 0.83 4.08 0.14 3.89 0.24 4.37 0.30 7 4.26 0.22 4.17 0.13 3.37 0.20 2.68 0.71 3.23 0.23 3.57 0.52 4.33 0.3310 4.38 0.17 4.09 0.14 3.12 0.17 2.60 0.61 2.90 0.30 3.66 0.53 4.43 0.2514 4.37 0.20 3.86 0.33 2.82 0.21 2.69 0.45 2.67 0.38 3.52 0.52 4.53 0.2217 4.47 0.12 4.10 0.23 3.03 0.20 2.91 0.36 2.20 0.84 3.62 0.49 3.71 1.5221 4.55 0.12 4.21 0.22 3.37 0.20 3.36 0.44 2.92 0.24 3.72 0.50 4.64 0.1124 4.57 0.11 4.36 0.20 3.67 0.08 3.91 0.34 3.21 0.25 4.06 0.32 4.72 0.15B Saline SEQID808 SEQID814 SEQID826 SEQID825 HBsAg HBsAg HBsAg HBsAgHBsAg Day stdev stdev stdev stdev stdev  0 4.68 0.10 4.64 0.05 4.67 0.074.00 0.43 4.50 0.09  3 4.40 0.12 3.99 0.09 3.64 0.16 3.21 0.90 3.44 0.05 6 4.49 0.09 3.56 0.07 2.78 0.20 2.62 1.03 2.59 0.13  9 4.50 0.07 3.240.07 2.36 0.16 2.32 1.00 2.16 0.20 13 4.71 0.07 3.16 0.14 2.29 0.15 2.211.03 2.03 0.22 16 4.58 0.05 3.10 0.15 2.64 0.08 2.37 1.05 2.27 0.21 204.71 0.03 3.37 0.21 3.05 0.14 2.45 1.18 2.47 0.33 23 4.62 0.08 3.47 0.233.36 0.12 2.68 1.24 2.54 0.62 C Saline SEQID824 HBsAg HBsAg day stdevstdev  0 4.49 0.13 4.67 0.03  3 4.62 0.13 4.14 0.06  6 4.50 0.14 3.000.13  9 4.45 0.21 2.35 0.15 13 4.36 0.38 2.19 0.25 16 4.20 0.66 2.450.20 20 4.46 0.09 3.45 0.22 23 3.97 1.05 2.42 0.18

From these data it can be concluded that in vivo all GalNAc conjugatedanti-HBV antisense oligomers are capable of reducing serum levels of HBVs antigen (HBsAg) to a level that is lower than saline and standard ofcare. In particular SEQ ID NO 807, SEQ ID NO: 808, SEQ ID NO: 814, SEQID NO: 815, SEQ ID NO: 825, SEQ ID NO: 826 can be demonstrated togreatly reduce the serum levels of HBsAg.

TABLE 8A-C Serum level of HBeAg (log₁₀(NCU/ml)) following twice-weeklydosages of 2 mg/kg A Saline SEQID806 SEQID807 SEQID815 SEQID800 SEQID802ETV HBeAg HBeAg HBeAg HBeAg HBeAg HBeAg HBeAg St St St St St St St Daydev dev dev dev dev dev dev  0 3.63 0.05 3.66 0.05 3.62 0.03 3.65 0.043.63 0.03 3.63 0.03 3.59 0.05  3 3.54 0.04 3.21 0.06 2.91 0.05 3.04 0.052.65 0.03 3.43 0.04 3.62 0.03  7 3.61 0.08 3.00 0.05 2.41 0.14 2.54 0.132.19 0.05 3.37 0.03 3.64 0.02 10 3.63 0.05 2.21 1.05 2.21 0.11 2.35 0.931.96 0.05 2.29 0.91 3.67 0.02 14 3.63 0.04 2.69 0.34 2.09 0.10 2.95 0.291.98 0.10 2.96 0.33 3.67 0.03 17 3.66 0.06 2.90 0.24 2.27 0.10 2.84 0.301.73 0.54 2.83 0.25 2.96 1.20 21 3.67 0.04 3.05 0.08 2.45 0.09 2.82 0.102.07 0.20 3.33 0.03 3.68 0.01 24 3.70 0.07 3.27 0.03 2.66 0.06 3.12 0.062.29 0.03 3.40 0.02 3.74 0.04 B Saline SEQID808 SEQID814 SEQID826SEQID825 HBeAg HBeAg HBeAg HBeAg HBeAg Day stdev stdev stdev stdev Stdev 0 3.72 0.08 3.63 0.03 3.56 0.03 3.32 0.17 3.51 0.12  3 3.43 0.06 2.740.03 2.48 0.08 2.82 0.18 2.31 0.07  6 3.44 0.02 2.35 0.06 1.98 0.07 2.430.16 1.94 0.06  9 3.36 0.02 2.03 0.11 1.84 0.07 2.22 0.11 1.76 0.05 133.72 0.05 2.25 0.08 1.96 0.03 2.23 0.09 1.95 0.08 16 3.66 0.10 2.32 0.062.08 0.04 2.45 0.15 1.98 0.08 20 3.74 0.01 2.58 0.04 2.36 0.05 2.67 0.182.21 0.11 23 3.65 0.05 2.60 0.07 2.52 0.08 2.82 0.10 2.33 0.11 C SalineSEQID824 HBeAg HBeAg day stdev stdev  0 3.75 0.02 3.74 0.02  3 3.52 0.092.35 0.07  6 3.45 0.06 1.89 0.03  9 3.60 0.06 1.94 0.04 13 3.58 0.101.58 0.04 16 3.58 0.15 1.64 0.04 20 3.48 0.13 2.69 0.05 23 3.59 0.121.75 0.02

From these data it can be concluded that in vivo all GalNAc conjugatedanti-HBV antisense oligomers are capable of reducing serum levels ofHBeAg to a level that is lower than saline and standard of care. Inparticular SEQ ID NO: 807, SEQ ID NO: 808, SEQ ID NO: 814, SEQ ID NO:815, SEQ ID NO: 825, SEQ ID NO: 826 can be demonstrated to greatlyreduce the serum levels of HBeAg.

TABLE 9A-C Serum level of HBV DNA (by log₁₀ copy number) followingbiweekly dosages of 2 mg/kg A Saline SEQID806 SEQID807 SEQID815 SEQID800SEQID802 ETV DNA DNA DNA DNA DNA DNA DNA Day StDev StDev StDev StDevStDev StDev StDev 0 7.08 0.54 7.78 0.08 7.27 0.38 7.06 0.39 7.26 0.356.92 0.31 7.30 0.37 3 7.21 0.36 7.28 0.20 6.45 0.54 6.11 0.41 5.99 0.456.78 0.33 5.73 0.83 7 6.81 0.54 6.53 0.15 5.16 0.86 4.52 0.38 5.16 0.506.43 0.58 5.15 0.50 10 7.64 0.14 6.38 0.37 4.72 0.52 4.23 LLOQ 4.23 LLOQ6.39 0.60 4.23 LLOQ 14 7.71 0.09 5.97 0.70 4.23 LLOQ 4.23 LLOQ 4.23 LLOQ6.12 0.46 4.23 LLOQ 17 7.76 0.04 6.13 0.57 4.23 LLOQ 4.56 0.57 4.23 LLOQ6.07 0.58 4.23 LLOQ 21 7.80 0.08 6.62 0.43 4.29 0.11 4.65 0.49 4.44 0.356.54 0.42 4.89 0.41 24 8.01 0.03 6.91 0.34 4.65 0.41 5.60 0.68 4.23 LLOQ7.01 0.33 5.22 0.57 LLOQ = less than lower level of quantification BSaline SEQID808 SEQID814 SEQID826 SEQID825 DNA DNA DNA DNA DNA Day StDevStDev StDev StDev StDev 0 6.95 0.35 6.79 0.30 7.13 0.24 6.94 0.40 7.020.18 3 7.15 0.26 5.92 0.27 5.88 0.15 6.25 0.50 5.90 0.35 6 7.26 0.224.80 0.64 4.44 0.48 5.34 0.70 4.56 0.68 9 7.44 0.23 4.16 LLOQ 4.16 LLOQ4.78 0.62 4.16 LLOQ 13 7.13 0.26 4.16 LLOQ 4.16 LLOQ 4.35 0.33 4.16 LLOQ16 7.04 0.44 4.16 LLOQ 4.16 LLOQ 4.27 0.20 4.16 LLOQ 20 7.04 0.36 4.16LLOQ 4.16 LLOQ 4.40 0.41 4.16 LLOQ 23 7.24 0.14 4.16 LLOQ 4.16 LLOQ 4.770.63 4.34 0.30 LLOQ = less than lower level of quantification C SalineSEQID824 DNA DNA day StDev StDev 0 7.47 0.23 7.33 0.16 3 7.55 0.21 5.990.25 6 7.74 0.19 4.89 0.48 9 7.76 0.21 4.51 0.40 13 7.82 0.27 4.32 LLOQ16 7.60 0.42 4.32 LLOQ 20 7.42 0.16 5.03 0.53 23 7.58 0.57 4.32 LLOQLLOQ = less than lower level of quantification

From these data it can be concluded that all GalNAc conjugated anti-HBVantisense oligomers are capable of reducing serum levels of HBV genomicDNA to a level that is lower than saline and or equal to the ETV (theclinical standard of care). In particular SEQ ID NO: 807, SEQ ID NO:808, SEQ ID NO: 814, SEQ ID NO: 815, SEQ ID NO: 825, SEQ ID NO: 826 canbe demonstrated to greatly reduce the serum levels of HBV genomic DNA.

The overall conclusion from these data is that in vivo theGalNAc-conjugated, HBV-targeting LNAs can target and reduce expressionof HBsAg and HBeAg better than ETV (the clinical standard of care) andthe HBV serum DNA with equal or better efficacy as compared to ETV.Given the broader effect on the viral transcriptional program than thenucleoside analog ETV, these data suggest that the use of GalNAcconjugated HBV-targeting LNAs in the clinic is likely to lead to a muchimproved outcome, including significantly increasing the cure rate forchronically infected HBV patients. Particularly the reduction of theimmune suppressor HBsAg will lead to a recovery of the HBV-directed hostimmune response.

Example 5 In Vivo Study of Bi-Weekly Injections at Several Doses

The AAV/HBV Mouse Model as prepared in Example 3 was used in this study.Seven GalNAc conjugated Anti-HBV LNA oligomers were tested at differentdoses with saline as control in C57BL/6 mice with stable viremia.

Mice were dosed subcutaneously twice weekly for two weeks on days 0, 3,7 and 10 or day 0, 3, 6 and 9 with the dose in mg/kg (mpk) pr injectionindicated in the tables below. HBV surface antigen (HBsAg), HBV eantigen (HBeAg), and HBV genomic DNA in serum was measured at theindicated days using the methods described in the “Materials andmethods” section. The mice were followed for 23-24 days.

The results are shown in the tables below.

TABLE 10A-G Serum level of HBsAg (log₁₀(IU/ml)) following biweeklydosages at the concentration indicated A SEQID 807 Saline 7.1 mpk 1.4mpk 0.28 mpk HBsAg HBsAg HBsAg HBsAg Serum Serum Serum Serum Day levelstdev level stdev level stdev level stdev 0 4.63 0.12 4.74 0.06 4.710.05 4.51 0.24 3 4.67 0.09 3.79 0.19 4.23 0.08 4.23 0.45 6 4.63 0.112.68 0.15 3.50 0.08 3.83 0.52 9 4.62 0.10 2.09 0.09 2.95 0.05 3.62 0.4213 4.64 0.06 1.84 0.09 2.50 0.04 3.54 0.28 16 4.56 0.05 1.72 0.05 2.530.13 3.63 0.35 20 4.69 0.03 1.97 0.29 2.92 0.17 3.91 0.25 23 4.67 0.101.78 0.11 3.18 0.15 4.08 0.20 B SEQID815 Saline 7.5 mpk 1.5 mpk 0.3 mpkHBsAg HBsAg HBsAg HBsAg Serum Serum Serum Serum Day level stdev levelstdev level stdev level stdev 0 4.63 0.12 4.74 0.05 4.67 0.18 4.14 0.613 4.67 0.09 3.47 0.10 3.55 0.38 3.46 0.99 7 4.63 0.11 2.25 0.20 2.700.32 3.02 0.99 10 4.62 0.10 1.89 0.17 2.07 0.27 2.68 1.14 14 4.64 0.061.48 0.19 1.81 0.24 2.60 1.07 17 4.56 0.05 1.59 0.19 2.22 0.21 2.79 1.1321 4.69 0.03 1.68 0.16 2.99 0.16 3.24 1.15 24 4.67 0.10 2.17 0.29 3.540.16 3.53 1.11 C SEQID814 Saline 6.15 mg/kg 1.26 mg/kg 0.252 mg/kg HBsAgHBsAg HBsAg HBsAg Serum Serum Serum Serum Day level stdev level stdevlevel stdev level stdev 0 4.49 0.13 4.59 0.07 4.55 0.17 4.48 0.18 3 4.620.13 3.56 0.21 4.02 0.29 4.51 0.18 7 4.5 0.14 2.22 0.23 3.26 0.23 4.250.23 10 4.45 0.21 1.89 0.24 2.9 0.2 4.21 0.22 14 4.36 0.38 1.69 0.272.77 0.24 4.33 0.16 17 4.2 0.66 1.75 0.2 3.02 0.19 4.4 0.09 21 4.46 0.092.13 0.21 3.5 0.29 4.31 0.17 24 3.97 1.05 2.45 0.17 3.54 0.36 4.51 0.09D SEQID825 Saline 7.5 mg/kg 1.5 mg/kg 0.3 mg/kg HBsAg HBsAg HBsAg HBsAgSerum Serum Serum Serum Day level stdev level stdev level stdev levelstdev 0 4.49 0.13 4.62 0.06 4.51 0.04 4.48 0.25 3 4.62 0.13 3.56 0.183.73 0.15 4.47 0.31 7 4.5 0.14 2.36 0.12 2.84 0.16 4.15 0.32 10 4.450.21 1.99 0.08 2.43 0.15 3.95 0.35 14 4.36 0.38 1.87 0.09 2.2 0.12 4.010.25 17 4.2 0.66 1.93 0.06 2.51 0.12 4.1 0.22 21 4.46 0.09 2.2 0.11 3.040.14 4.42 0.13 24 3.97 1.05 2.45 0.19 3.32 0.17 4.49 0.08 E SEQID808 7.1mg/ 1.42 mg/ 0.29 mg/ Saline kg SC kg SC kg SC HBsAg HBsAg HBsAg HBsAgSerum Serum Serum Serum Day level stdev level stdev level stdev levelstdev 0 4.78 0.11 4.58 0.29 4.86 0.08 4.55 0.34 3 4.75 0.1 3.38 0.724.27 0.24 4.36 0.43 7 4.85 0.05 2.8 0.55 3.7 0.45 4.25 0.37 10 4.81 0.082.31 0.43 3.42 0.35 4.06 0.5 14 4.99 0.02 2.36 0.31 3.51 0.4 4.18 0.6817 4.91 0.04 2.36 0.31 3.52 0.3 4.11 0.68 21 4.89 0.04 2.26 0.34 3.660.35 4.32 0.64 24 4.8 0.06 2.4 0.28 3.87 0.3 4.45 0.47 F SEQID824 Saline7.4 mg/kg SC 1.5 mg/kg SC 0.3 mg/kg SC HBsAg HBsAg HBsAg HBsAg SerumSerum Serum Serum Day level stdev level stdev level stdev level stdev 04.78 0.11 4.78 0.05 4.57 0.34 4.7 0.11 3 4.75 0.1 3.86 0.23 4 0.51 4.60.15 7 4.85 0.05 2.44 0.32 2.87 0.54 4.15 0.16 10 4.81 0.08 2.38 0.252.18 0.58 3.86 0.17 14 4.99 0.02 2.85 0.4 2.21 0.82 3.84 0.28 17 4.910.04 2.85 0.41 2.28 0.82 3.49 0.54 21 4.89 0.04 2.25 0.91 3.63 0.74 244.8 0.06 2.2 0.76 3.7 0.66 G SEQID826 Saline 7.1 mg/kg 1.42 mg/kg 0.29mg/kg HBsAg HBsAg HBsAg HBsAg Serum Serum Serum Serum Day level stdevlevel stdev level stdev level stdev 0 4.78 0.11 4.61 0.26 4.67 0.06 4.570.14 3 4.75 0.1 3.78 0.45 4.42 0.03 4.66 0.2 7 4.85 0.05 2.46 0.51 3.840.13 4.52 0.2 10 4.81 0.08 2.02 0.57 3.61 0.11 4.42 0.24 14 4.99 0.022.08 0.63 3.65 0.27 4.63 0.21 17 4.91 0.04 1.94 0.55 3.73 0.18 4.57 0.2121 4.89 0.04 2.54 0.25 4.09 0.13 4.73 0.2 24 4.8 0.06 3.04 0.21 4.230.17 4.79 0.24

The above data are also presented in FIG. 11 .

TABLE 11A-G Serum level of HBeAg (log₁₀(NCU/ml)) following biweeklydosages at the concentrations indicated. A SEQID 807 Saline 7.1 mpk 1.4mpk 0.28 mpk HBeAg HBeAg HBeAg HBeAg Serum Serum Serum Serum Day levelstdev level stdev level stdev level stdev 0 3.83 0.04 3.82 0.02 3.780.02 3.75 0.03 3 3.74 0.02 2.24 0.04 2.83 0.07 3.39 0.05 6 3.69 0.021.67 0.04 2.32 0.06 3.16 0.03 9 3.68 0.03 1.48 0.05 2.04 0.03 3.01 0.0213 3.66 0.03 1.53 0.02 1.75 0.03 2.83 0.14 16 3.69 0.03 1.21 0.07 1.850.03 2.98 0.04 20 3.66 0.04 1.48 0.07 1.98 0.05 3.12 0.04 23 3.63 0.041.34 0.09 2.19 0.06 3.24 0.07 B SEQID815 Saline 7.5 mpk 1.5 mpk 0.3 mpkHBeAg HBeAg HBeAg HBeAg Serum Serum Serum Serum Day level stdev levelstdev level stdev level stdev 0 3.83 0.04 3.80 0.07 3.77 0.03 3.67 0.153 3.74 0.02 2.00 0.08 2.56 0.12 3.37 0.15 7 3.69 0.02 1.60 0.05 2.020.08 3.04 0.11 10 3.68 0.03 1.47 0.03 1.85 0.10 2.75 0.38 14 3.66 0.031.40 0.39 1.64 0.09 2.81 0.10 17 3.69 0.03 1.29 0.12 1.85 0.09 2.78 0.2921 3.66 0.04 1.74 0.14 2.27 0.09 3.10 0.23 24 3.63 0.04 1.88 0.07 2.500.09 3.19 0.18 C SEQID814 Saline 6.15 mg/kg 1.26 mg/kg 0.252 mg/kg HBeAgHBeAg HBeAg HBeAg Serum Serum Serum Serum Day level stdev level stdevlevel stdev level stdev 0 3.75 0.02 3.8 0.08 3.8 0.05 3.75 0.08 3 3.520.09 2.15 0.06 2.97 0.05 3.46 0.09 7 3.45 0.06 1.73 0.05 2.47 0.02 3.220.09 10 3.6 0.06 1.7 0.07 2.32 0.05 3.2 0.08 14 3.58 0.1 1.4 0.05 2.150.04 3.24 0.12 17 3.58 0.15 1.55 0.05 2.44 0.06 3.34 0.1 21 3.48 0.131.75 0.04 2.7 0.05 3.34 0.15 24 3.59 0.12 1.92 0.05 2.8 0.04 3.44 0.13 DSEQID825 Saline 7.5 mg/kg 1.5 mg/kg 0.3 mg/kg HBeAg HBeAg HBeAg HBeAgSerum Serum Serum Serum Day level stdev level stdev level stdev levelstdev 0 3.75 0.02 3.72 0.08 3.71 0.09 3.93 0.16 3 3.52 0.09 2.22 0.12.68 0.06 3.39 0.06 7 3.45 0.06 1.77 0.1 2.36 0.05 3.14 0.12 10 3.6 0.061.7 0.05 2.18 0.1 3.17 0.1 14 3.58 0.1 1.47 0.07 1.92 0.07 3.14 0.02 173.58 0.15 1.69 0.06 2.17 0.13 3.25 0.04 21 3.48 0.13 1.87 0.08 2.4 0.083.38 0.05 24 3.59 0.12 1.98 0.09 2.55 0.06 3.48 0.02 E SEQID808 Saline7.1 mg/kg SC 1.42 mg/kg SC 0.29 mg/kg SC HBeAg HBeAg HBeAg HBeAg SerumSerum Serum Serum Day level stdev level stdev level stdev level stdev 03.59 0.06 3.51 0.04 3.56 0.01 3.47 0.07 3 3.59 0.03 2.5 0.06 2.98 0.023.4 0.07 7 3.69 0.02 2.05 0.08 2.63 0.03 3.2 0.07 10 3.67 0.04 1.84 0.112.46 0.04 3.19 0.06 14 3.81 0.03 1.72 0.07 2.45 0.02 3.27 0.07 17 3.740.03 1.68 0.14 2.5 0.02 3.27 0.07 21 3.72 0.03 1.58 0.18 2.64 0.04 3.350.08 24 3.73 0.06 1.77 0.19 2.8 0.04 3.55 0.05 F SEQID824 Saline 7.4mg/kg SC 1.5 mg/kg SC 0.3 mg/kg SC HBeAg HBeAg HBeAg HBeAg Serum SerumSerum Serum Day level stdev level stdev level stdev level stdev 0 3.590.06 3.5 0.03 3.51 0.02 3.49 0.03 3 3.59 0.03 2.09 0.08 2.47 0.13 3.280.03 7 3.69 0.02 1.81 0.05 1.95 0.05 2.88 0.04 10 3.67 0.04 1.88 0.071.88 0.03 2.65 0.06 14 3.81 0.03 1.71 0.06 1.72 0.1 2.48 0.05 17 3.740.03 1.68 0.06 1.68 0.05 2.56 0.05 21 3.72 0.03 1.8 0.04 2.63 0.08 243.73 0.06 2.02 0.06 2.81 0.05 G SEQID826 Saline 7.1 mg/kg 1.42 mg/kg0.29 mg/kg HBeAg HBeAg HBeAg HBeAg Serum Serum Serum Serum Day levelstdev level stdev level stdev level stdev 0 3.59 0.06 3.44 0.09 3.450.06 3.5 0.02 3 3.59 0.03 2.58 0.08 3.17 0.07 3.52 0.02 7 3.69 0.02 1.810.08 2.8 0.1 3.4 0.03 10 3.67 0.04 1.45 0.06 2.67 0.09 3.37 0.05 14 3.810.03 1.27 0.05 2.54 0.12 3.39 0.07 17 3.74 0.03 1.51 0.05 2.76 0.08 3.450.04 21 3.72 0.03 1.83 0.11 2.9 0.08 3.54 0.03 24 3.73 0.06 2.23 0.183.18 0.1 3.66 0.03

The above data are also presented in FIG. 12 .

TABLE 12A-G Serum level of log₁₀(HBV DNA) (by copy number) followingbiweekly dosages at the concentrations indicated. A SEQID 807 Saline 7.1mpk 1.4 mpk 0.28 mpk DNA DNA DNA DNA Serum Serum Serum Serum Day levelStDev level StDev level StDev level StDev 0 6.64 0.31 6.77 0.57 6.460.22 6.80 0.38 3 6.58 0.42 4.86 0.58 4.61 0.49 6.10 0.11 6 7.25 0.494.43 0.18 4.32 LLOQ 5.87 0.31 9 7.14 0.23 4.32 LLOQ 4.32 0.01 5.19 0.5813 7.32 0.33 4.30 LLOQ 4.30 LLOQ 5.23 0.54 16 7.28 0.27 4.30 LLOQ 4.30LLOQ 5.60 0.27 20 7.23 0.31 4.30 LLOQ 4.40 0.17 6.05 0.19 23 7.43 0.284.30 LLOQ 4.30 LLOQ 6.41 0.20 LLOQ = less than lower level ofquantification B SEQID815 Saline 7.5 mpk 1.5 mpk 0.3 mpk DNA DNA DNA DNASerum Serum Serum Serum Day level StDev level StDev level StDev levelStDev 0 6.64 0.31 6.64 0.32 6.48 0.31 6.54 0.42 3 6.58 0.42 4.44 0.214.59 0.46 4.95 0.67 7 7.25 0.49 4.32 LLOQ 4.32 LLOQ 4.56 0.41 10 7.140.23 4.31 0.01 4.32 LLOQ 4.38 0.11 14 7.32 0.33 4.30 LLOQ 4.30 LLOQ 4.30LLOQ 17 7.28 0.27 4.30 LLOQ 4.30 LLOQ 4.64 0.58 21 7.23 0.31 4.30 LLOQ4.30 LLOQ 5.12 0.87 24 7.43 0.28 4.30 LLOQ 4.30 LLOQ 5.37 1.08 LLOQ =less than lower level of quantification C SEQID814 Saline 6.15 mg/kg1.26 mg/kg 0.252 mg/kg DNA DNA DNA DNA Serum Serum Serum Serum Day levelStDev level StDev level StDev level StDev 0 7.47 0.23 7.55 0.18 7.480.22 7.64 0.17 3 7.55 0.21 6.2 0.18 6.44 0.26 7.37 0.14 7 7.74 0.19 5.310.25 5.53 0.36 7.31 0.08 10 7.76 0.21 4.46 0.19 4.65 0.49 7.22 0.1 147.82 0.27 4.32 0 4.32 0 7.27 0.09 17 7.6 0.42 4.32 0.01 4.38 0.09 7.410.14 21 7.42 0.16 4.32 0 4.62 0.56 7.42 0.09 24 7.58 0.57 4.32 0 5.480.7 7.66 0.16 D SEQID825 Saline 7.5 mg/kg 1.5 mg/kg 0.3 mg/kg DNA DNADNA DNA Serum Serum Serum Serum Day level StDev level StDev level StDevlevel StDev 0 7.47 0.23 7.36 0.28 7.41 0.32 7.5 0.44 3 7.55 0.21 5.890.31 5.84 0.67 6.95 0.43 7 7.74 0.19 4.75 0.57 4.92 0.55 6.73 0.43 107.76 0.21 4.35 0.13 4.28 0 6.49 0.44 14 7.82 0.27 4.32 0 4.32 0 6.520.48 17 7.6 0.42 4.32 0.01 4.31 0.01 6.68 0.26 21 7.42 0.16 4.32 0 4.320 6.82 0.57 24 7.58 0.57 4.32 0.01 4.63 0.31 7.19 0.32 E SEQID808 Saline7.1 mg/kg SC 1.42 mg/kg SC 0.29 mg/kg SC DNA DNA DNA DNA Serum SerumSerum Serum Day level StDev level StDev level StDev level StDev 0 7.770.17 7.97 0.26 7.93 0.11 7.93 0.24 3 7.8 0.05 6.29 0.4 6.69 0.21 7.310.35 7 7.75 0.15 5.02 0.59 5.67 0.43 7.24 0.35 10 7.79 0.09 4.5 0.364.95 0.75 6.98 0.3 14 8.01 0.09 4.29 0 4.56 0.46 6.83 0.55 17 7.89 0.134.29 0 4.29 0 6.97 0.46 21 7.94 0.06 4.29 0 4.72 0.47 7.13 0.41 24 7.830.08 4.29 0 4.97 0.45 7.37 0.36 F SEQID824 Saline 7.4 mg/kg SC 1.5 mg/kgSC 0.3 mg/kg SC DNA DNA DNA DNA Serum Serum Serum Serum Day level StDevlevel StDev level StDev level StDev 0 7.77 0.17 8 0.24 7.98 0.23 7.870.17 3 7.8 0.05 6.64 0.22 6.44 0.47 7.05 0.24 7 7.75 0.15 5.58 0.32 5.340.75 6.25 0.26 10 7.79 0.09 4.59 0.36 4.87 0.4 5.64 0.3 14 8.01 0.094.29 0 4.59 0.2 4.81 0.35 17 7.89 0.13 4.29 0 4.29 0 4.61 0.37 21 7.940.06 4.29 0 4.94 0.43 24 7.83 0.08 4.29 0 5.24 0.56 G SEQID826 Saline7.1 mg/kg 1.42 mg/kg 0.29 mg/kg DNA DNA DNA DNA Serum Serum Serum SerumDay level StDev level StDev level StDev level StDev 0 7.77 0.17 7.860.29 7.86 0.18 7.87 0.22 3 7.8 0.05 6.45 0.24 7.05 0.34 7.72 0.18 7 7.750.15 5.32 0.47 6.42 0.31 7.57 0.16 10 7.79 0.09 4.76 0.47 6.01 0.48 7.530.12 14 8.01 0.09 4.29 0 5.39 0.7 7.53 0.14 17 7.89 0.13 4.29 0 5.470.83 7.57 0.16 21 7.94 0.06 4.29 0 6.24 0.5 7.66 0.16 24 7.83 0.08 4.290 6.43 0.55 7.74 0.16

The above data are also presented in FIG. 13 .

From these data it can be concluded that all GalNAc conjugated anti-HBVantisense oligomers are capable of reducing serum levels of HBsAG, HBeAGand HBV genomic DNA to a level that is lower than saline. In particularSEQ ID NO: 807, 814, 815 and 825 show very efficient HBsAG decrease evenat the intermediate dose. At the highest dose the antigen reduction ismaintained at least 11 days after treatment ended for SEQ ID NO: 807 and815. SEQ ID 814, 815 and 825 demonstrate the most efficacious knock-downof viral serum DNA, demonstrating a particularly potent effect on theviral polymerase-expressing transcripts.

Example 6 Comparing Antiviral Efficacy by Different Route ofAdministrations

The AAV/HBV Mouse Model as prepared in Example 3 was used in this study.A GalNAc conjugated Anti-HBV LNA oligomer was tested at different dosesusing either subcutaneous (SC) or intravenous (IV) administration routeswith saline as control in C57BL/6 mice with stable viremia.

Mice were dosed subcutaneously or intravenously twice weekly for twoweeks on days 0, 3, 6 and 9 with the dose in mg/kg (mpk) pr injectionindicated in the tables below. HBV surface antigen (HBsAg), HBV eantigen (HBeAg), and HBV genomic DNA in serum was measured at theindicated days using the methods described in the “Materials andmethods” section. The mice were followed for 23 days after first dosing.

The results are shown in the tables below.

TABLE 13A Serum level of HBsAg (log₁₀(IU/ml)) following biweekly SCdosages at the concentration indicated A SEQID 807 Saline 0.2 mpk 1.0mpk 5.0 mpk HBsAg HBsAg HBsAg HBsAg Serum Serum Serum Serum Day levelStDev level StDev level StDev level StDev 0 4.64 0.11 4.55 0.08 4.460.13 4.69 0.07 3 4.61 0.11 4.44 0.06 3.80 0.22 3.64 0.10 6 4.58 0.074.29 0.08 3.33 0.19 2.54 0.12 9 4.57 0.10 4.11 0.05 2.98 0.20 1.83 0.1413 4.67 0.08 3.93 0.18 2.85 0.23 2.00 0.08 16 4.61 0.08 3.98 0.15 2.930.19 2.05 0.09 20 4.52 0.08 4.08 0.11 3.11 0.31 1.98 0.11 23 4.25 0.523.97 0.26 3.30 0.33 2.05 0.16

TABLE 13B Serum level of HBsAg (log₁₀(IU/ml)) following biweekly IVdosages at the concentration indicated B SEQID 807 Saline 0.2 mpk 1.0mpk 5.0 mpk HBsAg HBsAg HBsAg HBsAg Serum Serum Serum Serum Day levelStDev level StDev level StDev level StDev 0 4.64 0.11 4.61 0.09 4.600.11 4.63 0.12 3 4.61 0.11 4.39 0.06 4.19 0.12 3.93 0.11 6 4.58 0.074.13 0.05 3.69 0.13 3.17 0.13 9 4.57 0.10 3.91 0.12 3.36 0.15 2.73 0.2013 4.67 0.08 3.72 0.21 3.16 0.02 2.36 0.14 16 4.61 0.08 3.70 0.30 3.140.14 2.47 0.16 20 4.52 0.08 3.86 0.28 3.31 0.14 2.57 0.12 23 4.25 0.523.99 0.34 3.59 0.13 2.86 0.11

TABLE 14A Serum level of HBeAg (log₁₀(IU/ml)) following biweekly SCdosages at the concentration indicated. A SEQID 807 Saline 0.2 mpk 1.0mpk 5.0 mpk HBeAg HBeAg HBeAg HBeAg Serum Serum Serum Serum Day levelStDev level StDev level StDev level StDev 0 3.57 0.06 3.57 0.04 3.570.04 6.85 0.60 3 3.63 0.04 3.43 0.03 2.87 0.07 5.74 0.82 6 3.61 0.043.28 0.03 2.42 0.09 5.27 0.60 9 3.63 0.05 3.15 0.03 2.14 0.09 4.57 0.4013 3.49 0.07 2.84 0.09 2.01 0.05 4.30 LLOQ 16 3.53 0.06 3.06 0.04 2.090.06 4.30 LLOQ 20 3.56 0.05 3.21 0.03 2.24 0.08 4.30 LLOQ 23 3.64 0.063.29 0.03 2.46 0.09 4.85 0.54 LLOQ = less than lower level ofquantification

TABLE 14B Serum level of HBeAg (log₁₀(IU/ml)) following biweekly IVdosages at the concentration indicated B SEQID 807 Saline 0.2 mpk 1.0mpk 5.0 mpk HBeAg HBeAg HBeAg HBeAg Serum Serum Serum Serum Day levelStDev level StDev level StDev level StDev 0 3.57 0.06 3.65 0.06 3.620.04 3.61 0.08 3 3.63 0.04 2.13 0.06 3.37 0.05 2.96 0.03 6 3.61 0.041.59 0.05 3.05 0.04 2.52 0.07 9 3.63 0.05 1.39 0.07 2.88 0.06 2.35 0.0313 3.49 0.07 1.55 0.03 2.63 0.04 2.10 0.04 16 3.53 0.06 1.59 0.04 2.730.06 2.18 0.08 20 3.56 0.05 1.60 0.03 3.01 0.06 2.37 0.11 23 3.64 0.061.64 0.08 3.22 0.05 2.60 0.07

TABLE 15A Serum level of HBV DNA (by log₁₀ (copy number)) followingbiweekly SC dosages at the concentrations indicated A SEQID 807 Saline0.2 mpk 1.0 mpk 5.0 mpk DNA DNA DNA DNA Serum Serum Serum Serum Daylevel StDev level StDev level StDev level StDev 0 6.50 0.21 6.56 0.196.85 0.60 6.48 0.14 3 6.65 0.31 6.30 0.33 5.74 0.82 4.34 LLOQ 6 6.830.31 6.20 0.33 5.27 0.60 4.34 LLOQ 9 6.94 0.37 5.90 0.32 4.57 0.40 4.730.67 13 7.13 0.21 5.82 0.88 4.30 LLOQ 4.56 0.46 16 7.12 0.31 6.23 0.384.30 LLOQ 4.30 LLOQ 20 7.06 0.17 6.27 0.35 4.30 LLOQ 4.30 LLOQ 23 6.940.32 6.42 0.32 4.85 0.54 4.30 LLOQ LLOQ = less than lower level ofquantification

TABLE 15B Serum level of HBV DNA (by log₁₀ (copy number)) followingbiweekly IV dosages at the concentrations indicated A SEQID 807 Saline0.2 mpk 1.0 mpk 5.0 mpk DNA DNA DNA DNA Serum Serum Serum Serum Daylevel StDev level StDev level StDev level StDev 0 6.50 0.21 6.71 0.596.62 0.23 6.71 0.45 3 6.65 0.31 6.20 0.59 5.50 0.43 5.24 0.69 6 6.830.31 5.50 0.90 4.62 0.48 4.34 LLOQ 9 6.94 0.37 5.18 0.85 4.34 LLOQ 4.34LLOQ 13 7.13 0.21 5.05 0.76 4.30 LLOQ 4.30 LLOQ 16 7.12 0.31 5.13 0.844.30 LLOQ 4.30 LLOQ 20 7.06 0.17 5.50 0.80 4.30 LLOQ 4.30 LLOQ 23 6.940.32 5.99 0.41 4.30 LLOQ 4.30 LLOQ LLOQ = less than lower level ofquantification

The data are also presented in FIG. 14 .

From these data it can be concluded that administration of GalNAcconjugated anti-HBV antisense oligomers more efficiently reduces serumlevels of HBsAG, HBeAG and HBV genomic DNA to a level that is lower thansaline, when dosed by subcutaneous administration, as compared tointravenous administration.

Example 7: Comparison of Conjugated and Unconjugated Oligonucleotides

The AAV/HBV Mouse Model as prepared in Example 3 was used in this study.Unconjugated (SEQ ID NO 308 and 303) and GalNAc conjugated (SEQ ID NO:807 and 815) Anti-HBV LNA oligomers were tested at equimolar oligomerdoses with saline as control in C57BL/6 mice with stable viremia.

Mice were dosed subcutaneously twice weekly for two weeks on days 0, 3,6 and 9 with the dose in mg/kg (mpk) pr injection indicated in thetables below. HBV surface antigen (HBsAg), HBV e antigen (HBeAg), andHBV genomic DNA in serum was measured at the indicated days using themethods described in the “Materials and methods” section. The mice werefollowed for 23.

The results are shown in the tables below.

TABLE 16A-B Serum level of HBsAg (log₁₀(IU/ml)) following biweeklydosages at equimolarconcentrations A SEQ ID 308 SEQ ID 807 Saline 5 mpk7.1 mpk HBsAg HBsAg HBsAg Serum Serum Serum Day level StDev level StDevlevel StDev 0 4.63 0.12 4.70 0.05 4.74 0.06 3 4.67 0.09 4.44 0.14 3.790.19 6 4.63 0.11 3.91 0.44 2.68 0.15 9 4.62 0.10 3.50 0.44 2.09 0.09 134.64 0.06 3.27 0.43 1.84 0.09 16 4.56 0.05 3.20 0.53 1.72 0.05 20 4.690.03 3.61 0.54 1.97 0.29 23 4.67 0.10 3.96 0.34 1.78 0.11 B SEQ ID 303SEQ ID 815 Saline 1 mpk 1.5 mpk HBsAg HBsAg HBsAg Serum Serum Serum Daylevel StDev level StDev level StDev 0 4.63 0.28 4.49 0.54 4.62 0.25 34.70 0.17 4.58 0.38 3.31 0.09 6 4.84 0.12 4.78 0.17 3.21 0.14 9 4.810.13 4.81 0.13 2.64 0.22 13 4.86 0.12 4.91 0.06 2.63 0.58 16 4.83 0.134.85 0.10 2.04 0.57 20 4.73 0.18 4.68 0.14 2.26 0.58 23 4.66 0.20 4.480.40 2.74 0.64

TABLE 17A-B Serum level of HBeAg (log₁₀(IU/ml)) following biweeklydosages at equimolar concentrations A SEQ ID 308 SEQ ID 807 Saline 5 mpk7.1 mpk HBeAg HBeAg HBeAg Serum Serum Serum Day level StDev level StDevlevel StDev 0 3.83 0.04 3.79 0.05 3.82 0.02 3 3.74 0.02 3.39 0.08 2.240.04 6 3.69 0.02 3.07 0.02 1.67 0.04 9 3.68 0.03 2.82 0.04 1.48 0.05 133.66 0.03 2.66 0.03 1.53 0.02 16 3.69 0.03 2.75 0.03 1.21 0.07 20 3.660.04 3.01 0.02 1.48 0.07 23 3.63 0.04 3.10 0.05 1.34 0.09 B SEQ ID 303SEQ ID 815 Saline 1 mpk 1.5 mpk HBeAg HBeAg HBeAg Serum Serum Serum Daylevel StDev level StDev level StDev 0 3.82 0.03 3.74 0.04 3.80 0.06 33.82 0.03 3.78 0.02 2.48 0.10 6 3.85 0.02 3.83 0.06 2.70 0.05 9 3.810.03 3.75 0.02 2.29 0.07 13 3.84 0.02 3.84 0.03 2.45 0.08 16 3.84 0.043.80 0.02 2.19 0.02 20 3.78 0.04 3.73 0.04 2.27 0.06 23 3.79 0.03 3.790.01 2.60 0.04

TABLE 18A-B Serum level of HBV DNA (by log₁₀ (copy number)) followingbiweekly dosages at equimolar concentrations A SEQ ID 308 SEQ ID 807Saline DNA DNA DNA 5 mpk 7.1 mpk Serum Serum Serum Day level StDev levelStDev level StDev 0 6.64 0.31 6.53 0.26 6.77 0.57 3 6.58 0.42 5.18 0.884.86 0.58 6 7.25 0.49 5.05 0.73 4.43 0.18 9 7.14 0.23 4.54 0.37 4.32LLOQ 13 7.32 0.33 4.30 LLOQ 4.30 LLOQ 16 7.28 0.27 4.41 0.19 4.30 LLOQ20 7.23 0.31 4.63 0.57 4.30 LLOQ 23 7.43 0.28 5.23 0.93 4.30 LLOQ LLOQ =less than lower level of quantification B SEQ ID 303 SEQ ID 815 SalineDNA DNA DNA 1 mpk 1.5 mpk Serum Serum Serum Day level StDev level StDevlevel StDev 0 7.89 0.16 7.91 0.42 7.91 0.23 3 8.28 0.13 8.06 0.41 6.540.25 6 8.03 0.13 7.94 0.20 5.38 0.63 9 8.19 0.13 8.07 0.14 4.30 LLOQ 138.37 0.16 8.31 0.13 4.30 LLOQ 16 8.41 0.19 8.16 0.18 4.30 LLOQ 20 8.230.09 7.94 0.19 4.30 LLOQ 23 8.20 0.06 7.89 0.28 4.30 LLOQ LLOQ = lessthan lower level of quantification

The data are also presented in FIG. 15 .

From these data it can be concluded that administration of GalNAcconjugated anti-HBV antisense oligomers more efficiently reduces serumlevels of HBsAG and HBeAG to a level that is lower than saline, whendosed by subcutaneous administration, as compared to intravenousadministration. Serum HBV DNA is reduced with equal efficacy by the twomethods of delivery, although the limitation of the assay precludesdiscrimination at high doses.

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference in theirentirety and to the same extent as if each reference were individuallyand specifically indicated to be incorporated by reference and were setforth in its entirety herein (to the maximum extent permitted by law).All headings and sub-headings are used herein for convenience only andshould not be construed as limiting the invention in any way. The use ofany and all examples, or exemplary language (e.g., “such as”) providedherein, is intended merely to better illuminate the invention and doesnot pose a limitation on the scope of the invention unless otherwiseclaimed. No language in the specification should be construed asindicating any non-claimed element as essential to the practice of theinvention. The citation and incorporation of patent documents herein isdone for convenience only and does not reflect any view of the validity,patentability, and/or enforceability of such patent documents. Thisinvention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw.

The invention claimed is:
 1. An oligomer conjugate comprising: a) anoligomer that is 20 nucleotides in length, is perfectly complementary to20 contiguous nucleotides of SEQ ID NO: 3 and comprises a nucleotidesequence selected from the group consisting of SEQ ID NO: 24; 25; 26;27; 28; 183; 184; 185; 186; 187; 188; 189; 190; 191; 192; 193; 194; 195;196 and 197; and b) a carrier component comprising an asialoglycoproteinreceptor (ASGP-R) targeting moiety, wherein the carrier component iscovalently attached to the first oligomer.
 2. The oligomer conjugate ofclaim 1, wherein the first oligomer is a gapmer oligomer of W-X-Y,wherein X represents at least 6 contiguous 2′-deoxyribonucleotides andeach of W and Y independently represent at least one modifiednucleotides selected from the group consisting of: 2′-O-alkyl-RNA units,2′-amino-DNA units, 2′-fluoro-DNA units, LNA units, arabino nucleic acid(ANA) units, 2′-fluoro-ANA units, HNA units, intercalating nucleic acid(INA) units, 2′MOE units, ethylene nucleic acid (ENA) units, unlinkednucleic acid (UNA) units, tricyclo DNA units and cET-LNA units.
 3. Theoligomer conjugate of claim 2, wherein the modified nucleotides areselected from MOE or LNA units.
 4. The oligomer conjugate of claim 3,wherein the LNA units are selected form the group consisting ofβ-D-oxy-LNA, α-L-oxy-LNA, β-D-thio-LNA, β-D-ENA, and β-D amino.
 5. Theoligomer conjugate of claim 2, wherein W and Y independently consist of3, 4 or 5 2′-O-methoxyethylribose sugar (2′-MOE) or units and region Xconsists of 8, 9, 10, 11 or 12 2′-deoxyribonucleotides.
 6. The oligomerconjugate of claim 2, wherein regions W-X-Y have 5-10-5 or 4-12-4 units.7. The oligomer conjugate of claim 1, wherein said ASGP-R targetingmoiety is selected from the group consisting of galactose,galactosamine, N-formyl-galactosamine, N-acetylgalactosamine (GalNAc),N-propionyl-galactosamine, N-n-butanoyl-galactosamine,N-isobutanoylgalactose-amine or a cluster of any one or more thereof. 8.The oligomer conjugate of claim 1, wherein the carrier component is aGalNAc cluster comprising two to four terminal GalNAc moieties.
 9. Theoligomer conjugate of claim 8, wherein the GalNAc cluster is a trivalentGalNAc selected from the group consisting of


10. The oligomer conjugate of claim 1, wherein the carrier componentcomprises GalNAc2.
 11. The oligomer conjugate of claim 1, wherein thecovalent attachment comprises a phosophodiester nucleotide linkercomprising 2-5 phosphodiester linked DNA or RNA nucleosides.
 12. Theoligomer conjugate of claim 11, wherein the phosphodiester nucleotidelinker comprises the sequence CA.
 13. A pharmaceutical compositioncomprising the oligomer conjugate of claim 1 and a pharmaceuticallyacceptable carrier or excipient.